Midkine is a secreted protein that acts as a growth factor or cytokine involved in cell survival and inflammatory processes. It accumulates in amyloid plaques, which are hallmarks of Alzheimer\'s Disease (AD). The reproducibility of Midkine research would be enhanced if the community had access to well-characterized anti-Midkine antibodies. In this study, we characterized 8 commercial Midkine antibodies for Western blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in a knockout cell line and isogenic parental control. These studies are part of a larger, collaborative initiative seeking to address the antibody reproducibility issue 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.

Intact-mass spectrometry has huge potential for clinical application, as it enables both quantitative and qualitative analysis of intact proteins and possibly unlocks additional pathophysiological information via, e.g., detection of specific post-translational modifications (PTMs). Such valuable and clinically useful selectivity is typically lost during conventional bottom-up mass spectrometry. We demonstrate an innovative immunoprecipitation protein enrichment assay coupled to ultrahigh performance liquid chromatography quadrupole time-of-flight high resolution mass spectrometry (UPLC-QToF-HRMS) for the fast and simple identification of the protein tumor marker Neuron Specific Enolase Gamma (NSEγ) at low endogenous concentrations in human serum. Additionally, using the combination of immunoaffinity purification with intact mass spectrometry, the presence of NSEγ in an acetylated form in human serum was detected. This highlights the unique potential of immunoaffinity intact mass spectrometry in clinical diagnostics.

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.

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.

Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations are situated in the last exon and we previously demonstrated escape from nonsense-mediated decay by detecting mutant ADNP mRNA in patient blood. In this study, wild-type and ADNP mutants are investigated at the protein level and therefore optimal detection of the protein is required. Detection of ADNP by means of western blotting has been ambiguous with reported antibodies resulting in non-specific bands without unique ADNP signal. Validation of an N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials, resulting in a unique band signal around 150 kDa for ADNP, above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. Our antibody panel was subsequently tested by immunoblotting, comparing parental and homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines and showed disappearance of the 150 kDa signal, indicative for intact ADNP. By means of both a GFPSpark and Flag-tag N-terminally fused to a human ADNP expression vector, we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. Furthermore, we were also able to visualize endogenous ADNP with our C-terminal antibody panel in heterozygous cell lines carrying ADNP patient mutations, while the truncated ADNP mutants could only be detected with epitope-tag-specific antibodies, suggesting that addition of an epitope-tag possibly helps stabilizing the protein. However, western blotting of patient-derived hiPSCs, immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detect a native mutant ADNP protein. In addition, an N-terminal immunoprecipitation-competent ADNP antibody enriched truncating mutants in overexpression lysates, whereas implementation of the same method failed to enrich a possible native mutant protein in immortalized patient-derived lymphoblastoid cell lines. This study aims to shape awareness for critical assessment of mutant ADNP protein analysis in Helsmoortel-Van der Aa syndrome.

UNASSIGNED: Ubiquitination serves as a fundamental post-translational modification in numerous cellular events. Yet, its role in regulating corneal epithelial wound healing (CEWH) remains elusive. This study endeavored to determine the function and mechanism of ubiquitination in CEWH.
UNASSIGNED: Western blot and immunoprecipitation were used to discern ubiquitination alterations during CEWH in mice. Interventions, including neuronally expressed developmentally downregulated 4 (Nedd4) siRNA and proteasome/lysosome inhibitor, assessed their impact on CEWH. In vitro analyses, such as the scratch wound assay, MTS assay, and EdU staining, were conducted to gauge cell migration and proliferation in human corneal epithelial cells (HCECs). Moreover, transfection of miR-30/200 coupled with a luciferase activity assay ascertained their regulatory mechanism on Nedd4.
UNASSIGNED: Global ubiquitination levels were markedly increased during the mouse CEWH. Importantly, the application of either proteasomal or lysosomal inhibitors notably impeded the healing process both in vivo and in vitro. Furthermore, Nedd4 was identified as an essential E3 ligase for CEWH. Nedd4 expression was significantly upregulated during CEWH. In vivo studies revealed that downregulation of Nedd4 substantially delayed CEWH, whereas further investigations underscored its role in regulating cell proliferation and migration, through the Stat3 pathway by targeting phosphatase and tensin homolog (PTEN). Notably, our findings pinpointed miR-30/200 family members as direct regulators of Nedd4.
UNASSIGNED: Ubiquitination holds pivotal significance in orchestrating CEWH. The critical E3 ligase Nedd4, under the regulatory purview of miR-30 and miR-200, facilitates CEWH through PTEN-mediated Stat3 signaling. This revelation sheds light on a prospective therapeutic target within the realm of CEWH.

Lysosomes are critical for the sustenance of glioblastoma stem-like cells (GSCs) properties. We present a protocol to enrich and purify lysosomes from patient-derived GSCs in culture. We describe the steps required to stably express a tagged lysosomal protein in GSCs, mechanically lyse cells, magnetically immunopurify lysosomes, and qualitatively assess these organelles. We then detail the procedure for retrieving intact and purified lysosomes from GSCs. We also specify cell culture conditions, storage procedures, and sample preparation for immunoblotting. For complete details on the use and execution of this protocol, please refer to Maghe et al.1.

UNASSIGNED: In a patient with anti-aminoacyl tRNA synthetase antibody and anti-OJ antibody syndrome, interventions likes warming, prostaglandins, and antiplatelets failed. However, prednisolone pulse treatment rapidly halted disease progression. Patients with mild interstitial pneumonia, myositis, and extremity necrosis should be promptly considered for anti-synthetase syndrome and receive immunosuppression after ruling out other causes.
UNASSIGNED: Anti-aminoacyl tRNA synthetase (ARS) autoantibodies are myositis-specific, and patients who test positive for ARS and have common clinical features are usually diagnosed with antisynthetase antibody syndrome (antisynthetase syndrome). Anti-ARS antibodies include histidyl-tRNA synthetase-1 (Jo-1), anti-threonyl (PL-7), anti-alanyl (PL-12), anti-glycyl (EJ), anti-asparaginyl (KS), anti-tyrosyl (Ha), and anti-phenylalanyl (Zo) tRNA synthetases. Among these, anti-isoleucyl tRNA synthetase (OJ) autoantibodies are extremely rare, and patients with these are frequently complicated by interstitial pneumonia. We report the case of an older man with ARS antibody syndrome who tested positive for anti-OJ and anti-Sjögren\'s-syndrome-related antigen A (Ro-52) antibodies. He had muscle weakness due to myositis and unparalleled rapid and severe finger necrosis. Pulsed prednisolone effectively treated the myositis symptoms and terminated the progression of finger necrosis.

Proteins are often referred to as the workhorses of cells, and their interactions are necessary to facilitate specific cellular functions. Despite the recognition that protein-protein interactions, and thus protein functions, are determined by proteoform states, such as mutations and post-translational modifications (PTMs), methods for determining the differential abundance of proteoforms across conditions are very limited. Classically, immunoprecipitation coupled with mass spectrometry (IP-MS) has been used to understand how the interactome (preys) of a given protein (bait) changes between conditions to elicit specific cellular functions. Reversing this concept, we present here a new workflow for IP-MS data analysis that focuses on identifying the differential peptidoforms of the bait protein between conditions. This method can provide detailed information about specific bait proteoforms, potentially revealing pathogenic protein states that can be exploited for the development of targeted therapies.

Protein-specific antibodies are essential for various aspects of protein research, including detection, purification, and characterization. When specific antibodies are unavailable, protein tagging is a useful alternative. Small epitope tags, typically less than 10 amino acids, are widely used in protein research due to the simple modification through PCR and reduced impact on the target protein\'s function compared to larger tags. The 2B8 epitope tag (RDPLPFFPP), reported by us in a previous study, has high specificity and sensitivity to the corresponding antibody. However, when attached to the C-terminus of the target protein in immunoprecipitation experiments, we observed a decrease in detection signal with reduced immunity and low protein recovery. This phenomenon was not unique to 2B8 and was also observed with the commercially available Myc tag. Our study revealed that C-terminal tagging of small epitope tags requires the addition of more than one extra amino acid to enhance (restore) antibody immunities. Moreover, among the amino acids we tested, serine was the best for the 2B8 tag. Our findings demonstrated that the interaction between a small epitope and a corresponding paratope of an antibody requires an extra amino acid at the C-terminus of the epitope. This result is important for researchers planning studies on target proteins using small epitope tags.