Human/Mouse/Rat IGSF8/CD316 Antibody

Detection of Human and Mouse IGSF8/CD316 by Western Blot. Western blot shows lysates of SH-SY5Y human neuroblastoma cell line, DU145 human prostate carcinoma cell line, and bEnd.3 mouse endothelioma cell line. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human/Mouse/Rat IGSF8/CD316 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3117) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). Specific bands were detected for IGSF8/CD316 at approximately 70-80 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Rat IGSF8/CD316 by Western Blot. Western blot shows lysates of rat brain (hippocampus) tissue. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human/Mouse/Rat IGSF8/CD316 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3117) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for IGSF8/CD316 at approximately 70 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of IGSF8/CD316 in Neuro‑2A Mouse Cell Line by Flow Cytometry. Neuro-2A mouse neuroblastoma cell line was stained with Goat Anti-Human/Mouse/Rat IGSF8/CD316 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3117, filled histogram) or isotype control antibody (Catalog # AB-108-C, open histogram), followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107).

IGSF8/CD316 in Neuro‑2A Mouse Cell Line. IGSF8/CD316 was detected in immersion fixed Neuro-2A mouse neuroblastoma cell line using Goat Anti-Human/Mouse/Rat IGSF8/CD316 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3117) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counterstained with DAPI (blue). Specific staining was localized to cell surfaces. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Detection of Human, Mouse, and Rat IGSF8/CD316 by Simple Western^TM. Simple Western lane view shows lysates of human cerebellum tissue, human hippocampus tissue, Neuro-2A mouse neuroblastoma cell line, and rat hippocampus tissue, loaded at 0.2 mg/mL. A specific band was detected for IGSF8/CD316 at approximately 71-80 kDa (as indicated) using 20 µg/mL of Goat Anti-Human/Mouse IGSF8/CD316 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3117) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Human IGSF8/CD316 by Western Blot WGA-HRP identifies a number of EV-specific markers that are present regardless of oncogene status.(A) Matrix depicting samples analyzed during LFQ comparison–Control and Myc cells, as well as Control and Myc EVs. (B) Principle component analysis (PCA) of all four groups analyzed by LFQ. Component 1 (50.4%) and component 2 (15.8%) are graphed. (C) Functional annotation was performed for each gene cluster using DAVID Bioinformatics Resource 6.8 and the highest ranking annotation features for the EV-specific gene cluster are shown. (D) Heatmap of the 50 most upregulated proteins in either RWPE-1 cells or EVs. Proteins are listed in decreasing order of expression with the most highly expressed proteins in EVs on the far left and the most highly expressed proteins in cells on the far right. Averages from all four replicates of each sample type are graphed. Scale indicates intensity, defined as (LFQ Area−Mean LFQ Area)/Standard Deviation. Extracellular proteins with annotated transmembrane domains are bolded and annotated secreted proteins are italicized. (E) Table indicating fold-change of most differentially regulated proteins by LC-MS/MS for RWPE-1 EVs compared to parent cells. (F) Western blot showing the EV-specific marker ITIH4, IGSF8, and MFGE8. Mass spectrometry data is based on two biological and two technical replicates (N=4). Due to limited sample yield, one replicate was performed for the EV western blot. EV, extracellular vesicle; LFQ, label-free quantification.Figure 5—source data 1.Uncropped western blots.Figure 5—source data 2.Mass spectrometry analysis results table.Figure 5—source data 3.List of proteins comparing enriched targets (>2-fold) in Control EVs versus Control cells and Myc EVs versus Myc cells.Uncropped western blots.Mass spectrometry analysis results table.List of proteins comparing enriched targets (>2-fold) in Control EVs versus Control cells and Myc EVs versus Myc cells.Heatmap comparison of biological and technical replicates of RWPE-1 Control/Myc cells and EVs.Biological and technical replicates cluster together based on both oncogene status and compartment for EV or cell surface. Proteins with no area values were assigned an imputed value using Perseus. Heatmap clustering is based off of the Pearson correlation between all replicates on both columns and rows. Heatmap was produced using Morpheus, https://software.broadinstitute.org/morpheus. The first number following the sample name denotes the biological replicated and second number denotes the technical replicate. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/35257663), licensed under a CC-BY license. Not internally tested by R&D Systems.