Identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence.

Moesin is a cytoskeletal adaptor protein, involved in the modification of the actin cytoskeleton, with relevance to Alzheimer's Disease. Well characterized anti-Moesin antibodies would benefit the scientific community. In this study, we have characterized ten Moesin commercial antibodies in Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications.

Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, have demonstrated that: (i) more than 50% of all antibodies failed in one or more applications, (ii) yet, ~50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and (iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.

Commercially produced antibodies are essential research tools. Investigators at universities and pharmaceutical companies use them to study human proteins, which carry out all the functions of the cells. Scientists usually buy antibodies from commercial manufacturers who produce more than 6 million antibody products altogether. Yet many commercial antibodies do not work as advertised. They do not recognize their intended protein target or may flag untargeted proteins. Both can skew research results and make it challenging to reproduce scientific studies, which is vital to scientific integrity. Using ineffective commercial antibodies likely wastes $1 billion in research funding each year. Large-scale validation of commercial antibodies by an independent third party could reduce the waste and misinformation associated with using ineffective commercial antibodies. Previous research testing an antibody validation pipeline showed that a commercial antibody widely used in studies to detect a protein involved in amyotrophic lateral sclerosis did not work. Meanwhile, the best-performing commercial antibodies were not used in research. Testing commercial antibodies and making the resulting data available would help scientists identify the best study tools and improve research reliability. Ayoubi et al. collaborated with antibody manufacturers and organizations that produce genetic knock-out cell lines to develop a system validating the effectiveness of commercial antibodies. In the experiments, Ayoubi et al. tested 614 commercial antibodies intended to detect 65 proteins involved in neurologic diseases. An effective antibody was available for about two thirds of the 65 proteins. Yet, hundreds of the antibodies, including many used widely in studies, were ineffective. Manufacturers removed some underperforming antibodies from the market or altered their recommended uses based on these data. Ayoubi et al. shared the resulting data on Zenodo, a publicly available preprint database. The experiments suggest that 20-30% of protein studies use ineffective antibodies, indicating a substantial need for independent assessment of commercial antibodies. Ayoubi et al. demonstrated their side-by-side antibody comparison methods were an effective and efficient way of validating commercial antibodies. Using this approach to test commercial antibodies against all human proteins would cost about $50 million. But it could save much of the $1 billion wasted each year on research involving ineffective antibodies. Independent validation of commercial antibodies could also reduce wasted efforts by scientists using ineffective antibodies and improve the reliability of research results. It would also enable faster, more reliable research that may help scientists understand diseases and develop new therapies to improve patient's lives.

Identification of high-performing antibodies for Superoxide dismutase [Cu-Zn] 1 (SOD1) for use in Western blot, immunoprecipitation, and immunofluorescence.

Superoxide dismutase [Cu-Zn] 1 (SOD1), is an antioxidant enzyme encoded by the gene SOD1, responsible for regulating oxidative stress levels by sequestering free radicals. Identified as the first gene with mutations in Amyotrophic lateral sclerosis (ALS), SOD1 is a determinant for studying diseases of aging and neurodegeneration. With guidance on well-characterized anti-SOD1 antibodies, the reproducibility of SOD1 research would be enhanced. In this study, we characterized eleven SOD1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

The identification of high-performing antibodies for Coiled-coil-helix-coiled-coil-helix domain containing protein 10 (CHCHD10) for use in Western Blot, immunoprecipitation and immunofluorescence.

CHCHD10 is a mitochondrial protein, implicated in the regulation of mitochondrial morphology and cristae structure, as well as the maintenance of mitochondrial DNA integrity. Recently discovered to be associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in its mutant form, the scientific community would benefit from the availability of validated anti-CHCHD10 antibodies. In this study, we characterized four CHCHD10 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. As this study highlights high-performing antibodies for CHCHD10, we encourage readers to use it as a guide to select the most appropriate antibody for their specific needs.

The identification of high-performing antibodies for Charged multivesicular body protein 2b for use in Western Blot, immunoprecipitation and immunofluorescence.

Charged multivesicular body protein 2B is a subunit of the endosomal sorting complex required for transport III (ESRCT-III), a complex implicated in the lysosomal degradation pathway and formation of multivesicular bodies. Mutations to the CHMP2B gene can result in abnormal protein aggregates in neurons and is therefore predicted to be associated in neurodegenerative diseases, including across the ALS-FTD spectrum. Through our standardized experimental protocol which compares read-outs in knockout cell lines and isogenic parental controls, this study aims to enhance the reproducibility of research on this target by characterizing eight commercial antibodies against charged multivesicular body protein 2b using Western Blot, immunoprecipitation, and immunofluorescence. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications.

Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, demonstrates that: i) more than 50% of all antibodies failed in one or more tests, ii) yet, ~50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first such study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.

The identification of high-performing antibodies for Sequestosome-1 for use in Western blot, immunoprecipitation and immunofluorescence.

Sequestosome-1, encoded by the gene SQSTM1, functions as a bridge between ubiquitinated proteins and the proteasome or autophagosome, thereby regulating protein degradation pathways. Loss of Sequestosome-1 is hypothesized to enhance neurodegeneration progression in several diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal disorders (FTD). Sequestosome-1 reproducible research would be facilitated with the availability of well-characterized anti-Sequestosome-1 antibodies. In this study, we characterized seventeen Sequestosome-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Identification of high-performing antibodies for tyrosine-protein kinase SYK for use in Western Blot, immunoprecipitation and immunofluorescence.

Tyrosine-protein kinase SYK, encoded by the SYK gene, is a non-receptor type protein kinase which mediates immune signal transduction through immunoreceptors. Tyrosine-protein kinase SYK expression has been associated with the development of various inflammatory diseases, cancer and neurodegenerative conditions. The reproducibility of tyrosine-protein kinase SYK research would help elucidate the mechanism in which it causes neuroinflammation as well as its potential as a novel target to treat Alzheimer's disease. This would be facilitated with the availability of high-quality tyrosine-protein kinase SYK. In this study, we characterized thirteen tyrosine-protein kinase SYK commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence.

TBK1 is a serine-threonine protein kinase that has been linked to a number of diseases including amyotrophic lateral sclerosis and frontotemporal dementia. Reproducible research on TBK1 has been hampered by the lack of well characterized antibodies. In this study, we characterized 11 commercial antibodies for TBK1 for use in immunoblot, immunofluorescence and immunoprecipitation, using an isogeneic knock-out cell line as a control. We identify antibodies that appear specific for all three applications but invite the readers to interpret the present findings based on their own scientific expertise and use this report as a guide to select the most appropriate antibody for their specific needs.

The ARSACS disease protein sacsin controls lysosomal positioning and reformation by regulating microtubule dynamics.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay is a fatal brain disorder featuring cerebellar neurodegeneration leading to spasticity and ataxia. This disease is caused by mutations in the SACS gene that encodes sacsin, a massive 4579-amino acid protein with multiple modular domains. However, molecular details of the function of sacsin are not clear. Here, using live cell imaging and biochemistry, we demonstrate that sacsin binds to microtubules and regulates microtubule dynamics. Loss of sacsin function in various cell types, including knockdown and KO primary neurons and patient fibroblasts, leads to alterations in lysosomal transport, positioning, function, and reformation following autophagy. Each of these phenotypic changes is consistent with altered microtubule dynamics. We further show the effects of sacsin are mediated at least in part through interactions with JIP3, an adapter for microtubule motors. These data reveal a new function for sacsin that explains its previously reported roles and phenotypes.

Sample Preparation for Multicolor STED Microscopy.

Stimulated emission depletion (STED) microscopy is one of the optical superresolution microscopy (SRM) techniques, more recently also referred to as nanoscopy, that have risen to popularity among biologists during the past decade. These techniques keep pushing the physical boundaries of optical resolution toward the molecular scale. Thereby, they enable biologists to image cellular and tissue structures at a level of almost molecular detail that was previously only achievable using electron microscopy. All the while, they retain the advantages of light microscopy, in particular with regards to sample preparation and flexibility of imaging. Commercially available SRM setups have become more and more available and also increasingly sophisticated, both in terms of optical performance and, importantly, ease of use. Institutional microscopy core facilities now offer widespread access to this type of systems. However, the field has grown so rapidly, and keeps growing, that biologists can be easily overwhelmed by the multitude of available techniques and approaches. From this vast array of SRM modalities, STED stands out in one respect: it is essentially an extension to an advanced confocal microscope. Most experienced users of confocal microscopy will find the transition to STED microscopy relatively easy as compared with some other SRM techniques. This also applies to STED sample preparation. Nonetheless, because resolution in STED microscopy does not only depend on the wavelength of the incident light and the numerical aperture of the objective, but crucially also on the square root of the intensity of the depletion laser and, in general, on the photochemical interaction of the fluorophore with the depletion laser, some additional considerations are necessary in STED sample preparation. Here we describe the single color staining of the somatostatin receptor subtype 2A (SSTR2A) and dual color staining of the trans-Golgi-network protein TGN 38 and the t-SNARE syntaxin-6 for STED in the endocrine cell line AtT20 and STED imaging of the samples, providing the protocols in as general a form as possible. The protocols in this chapter are used in this way in an institutional microscopy core facility.

Ultradian Secretion of Growth Hormone in Mice: Linking Physiology With Changes in Synapse Parameters Using Super-Resolution Microscopy.

Neuroendocrine circuits are orchestrated by the pituitary gland in response to hypothalamic hormone-releasing and inhibiting factors to generate an ultradian and/or circadian rhythm of hormone secretion. However, mechanisms that govern this rhythmicity are not fully understood. It has been shown that synaptic transmission in the rodent hypothalamus undergoes cyclical changes in parallel with rhythmic hormone secretion and a growing body of evidence suggests that rapid rewiring of hypothalamic neurons may be the source of these changes. For decades, structural synaptic studies have been utilizing electron microscopy, which provides the resolution suitable for visualizing synapses. However, the small field of view, limited specificity and manual analysis susceptible to bias fuel the search for a more quantitative approach. Here, we apply the fluorescence super-resolution microscopy approach direct Stochastic Optical Reconstruction Microscopy (dSTORM) to quantify and structurally characterize excitatory and inhibitory synapses that contact growth hormone-releasing-hormone (GHRH) neurons during peak and trough values of growth hormone (GH) concentration in mice. This approach relies on a three-color immunofluorescence staining of GHRH and pre- and post-synaptic markers, and a quantitative analysis with a Density-Based Spatial Clustering of Applications with Noise (DBSCAN) algorithm. With this method we confirm our previous findings, using electron microscopy, of increased excitatory synaptic input to GHRH neurons during peak levels of GH. Additionally, we find a shift in synapse numbers during low GH levels, where more inhibitory synaptic inputs are detected. Lastly, we utilize dSTORM to study novel aspects of synaptic structure. We show that more excitatory (but not inhibitory) pre-synaptic clusters associate with excitatory post-synaptic clusters during peaks of GH secretion and that the numbers of post-synaptic clusters increase during high hormone levels. The results presented here provide an opportunity to highlight dSTORM as a valuable quantitative approach to study synaptic structure in the neuroendocrine circuit. Importantly, our analysis of GH circuitry sheds light on the potential mechanism that drives ultradian changes in synaptic transmission and possibly aids in GH pulse generation in mice.

Characterization of agonist-dependent somatostatin receptor subtype 2 trafficking in neuroendocrine cells.

BACKGROUND: Somatostatin (SOM) receptor subtype 2 (SSTR2) is the major receptor subtype mediating SOM effects throughout the neuraxis. We previously demonstrated that the non-selective agonist [D-Trp8]-SOM induces intracellular sequestration of SSTR2, whereas this receptor is maintained at the cell surface after treatment with the SSTR2-selective agonist L-779,976 in cells co-expressing SSTR2 and SSTR5. METHODS AND RESULTS: In this study, we knocked-out SSTR5 in AtT20 cells endogenously expressing both SSTR2 and SSTR5 and used immuno-labeling and confocal microscopy to investigate the effect of SSTR5 on regulation of SSTR2 trafficking. Our results indicate that unlike [D-Trp8]-SOM-induced intracellular sequestration, L-779,976 stimulation results in the maintenance of SSTR2 at the cell surface regardless of whether SSTR5 is present or not. We then examined the trafficking pathways of SSTR2 upon stimulation by either agonist. We found that both [D-Trp8]-SOM and L-779,976 induce SSTR2 internalization via transferrin-positive vesicles. However, SSTR2 internalized upon L-779,976 treatment undergoes rapid recycling to the plasma membrane, whereas receptors internalized by [D-Trp8]-SOM recycle slowly after washout of the agonist. Furthermore, [D-Trp8]-SOM stimulation induces degradation of a fraction of internalized SSTR2 whereas L-779,976-dependent, rapid SSTR2 recycling appears to protect internalized SSTR2 from degradation. In addition, Octreotide which has preferential SSTR2 affinity, induced differential effects on both SSTR2 trafficking and degradation. CONCLUSION: Our results indicate that the biased agonistic property of L-779,976 protects against SSTR2 surface depletion by rapidly initiating SSTR2 recycling while SSTR5 does not regulate L-779-976-dependent SSTR2 trafficking.

Regulated resurfacing of a somatostatin receptor storage compartment fine-tunes pituitary secretion.

The surfacing of the glucose transporter GLUT4 driven by insulin receptor activation provides the prototypic example of a homeostasis response dependent on mobilization of an intracellular storage compartment. Here, we generalize this concept to a G protein-coupled receptor, somatostatin receptor subtype 2 (SSTR2), in pituitary cells. Following internalization in corticotropes, SSTR2 moves to a juxtanuclear syntaxin-6-positive compartment, where it remains until the corticotropes are stimulated with corticotropin releasing factor (CRF), whereupon SSTR2 exits the compartment on syntaxin-6-positive vesicular/tubular carriers that depend on Rab10 for their fusion with the plasma membrane. As SSTR2 activation antagonizes CRF-mediated hormone release, this storage/resurfacing mechanism may allow for a physiological homeostatic feedback system. In fact, we find that SSTR2 moves from an intracellular compartment to the cell surface in pituitary gland somatotropes, concomitant with increasing levels of serum growth hormone (GH) during natural GH cycles. Our data thus provide a mechanism by which signaling-mediated plasma membrane resurfacing of SSTR2 can fine-tune pituitary hormone release.

Cystic fibrosis transmembrane conductance regulator dysfunction in VIP knockout mice.

Vasoactive intestinal peptide (VIP), a neuropeptide, controls multiple functions in exocrine tissues, including inflammation, and relaxation of airway and vascular smooth muscles, and regulates CFTR-dependent secretion, which contributes to mucus hydration and local innate defense of the lung. We had previously reported that VIP stimulates the VPAC1 receptor, PKCϵ signaling cascade, and increases CFTR stability and function at the apical membrane of airway epithelial cells by reducing its internalization rate. Moreover, prolonged VIP stimulation corrects the molecular defects associated with F508del, the most common CFTR mutation responsible for the genetic disease cystic fibrosis. In the present study, we have examined the impact of the absence of VIP on CFTR maturation, cellular localization, and function in vivo using VIP knockout mice. We have conducted pathological assessments and detected signs of lung and intestinal disease. Immunodetection methods have shown that the absence of VIP results in CFTR intracellular retention despite normal expression and maturation levels. A subsequent loss of CFTR-dependent chloride current was measured in functional assays with Ussing chamber analysis of the small intestine ex vivo, creating a cystic fibrosis-like condition. Interestingly, intraperitoneal administration of VIP corrected tissue abnormalities, close to the wild-type phenotype, as well as associated defects in the vital CFTR protein. The results show in vivo a primary role for VIP chronic exposure in CFTR membrane stability and function and confirm in vitro data.

VIP regulates CFTR membrane expression and function in Calu-3 cells by increasing its interaction with NHERF1 and P-ERM in a VPAC1- and PKCε-dependent manner.

Vasoactive intestinal peptide (VIP) is a topical airway gland secretagogue regulating fluid secretions, primarily by stimulating cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride secretion that contributes to the airways innate defense mechanism. We previously reported that prolonged VIP stimulation of pituitary adenylate cyclase-activating peptide receptors (VPAC1) in airway cells enhances CFTR function by increasing its membrane stability. In the present study, we identified the key effectors in the VIP signaling cascade in the human bronchial serous cell line Calu-3. Using immunocytochemistry and in situ proximity ligation assays, we found that VIP stimulation increased CFTR membrane localization by promoting its colocalization and interaction with the scaffolding protein Na(+)/H(+) exchange factor 1 (NHERF1), a PDZ protein known as a positive regulator for CFTR membrane localization. VIP stimulation also increased phosphorylation, by protein kinase Cε of the actin-binding protein complex ezrin/radixin/moesin (ERM) and its interaction with NHERF1 and CFTR complex. On the other hand, it reduced intracellular CFTR colocalization and interaction with CFTR associated ligand, another PDZ protein known to compete with NHERF1 for CFTR interaction, inducing cytoplasmic retention and lysosomal degradation. Reducing NHERF1 or ERM expression levels by specific siRNAs prevented the VIP effect on CFTR membrane stability. Furthermore, iodide efflux assays confirmed that NHERF1 and P-ERM are necessary for VIP regulation of the stability and sustained activity of membrane CFTR. This study shows the cellular mechanism by which prolonged VIP stimulation of airway epithelial cells regulates CFTR-dependent secretions.