Journal: bioRxiv

Article Title: Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies to self-antigens

doi: 10.1101/2023.03.20.533453

Figure Lengend Snippet: ( A ) Proteins that were immunoprecipitated by antibody 8-3 from lysates of MiaPaca2 cells were separated by SDS-PAGE and visualized by silver staining. Two bands were identified by mass spectrometry (red arrowheads). ( B ) MiaPaca2 cells were co-stained with 8-3, phalloidin, and an antibody specific for MYH10 after 30min treatment of the cells with DMSO or cytochalasin D. ( C ) Dot-blot assay is shown the reaction with F-actin or G-actin of 8-3, isotype control IgG, and a commercial actin specific antibody. ( D ) The binding of incremental concentrations of 8-3 to F-actin was measured by ELISA. ( E ) Proteins that were immunoprecipitated by recombinant antibody 19-3 from lysates of MiaPaca2 cells were separated by SDS-PAGE and visualized by silver staining. The proteins, RUVBL1 and RUVBL2, were identified by mass spectrometry. ( F ) MiaPaca2 cell were co-stained with 19-3 and an antibody specific for RUVBL2. ( G ) The binding of incremental concentrations of 19-3 to RUVBL2 was measured by ELISA. ( H ) Western blot analysis of recombinant antibody 15-7 detection of cytoplasmic proteins (Cyto) and mitochondria-enriched proteins (Mito) is shown. ( I ) Immunofluorescent staining of MiaPaca2 cells by 15-7 and an antibody specific for HSPD1 is shown. ( J ) The binding of incremental concentrations of 15-7 to HSPD1 was measured by ELISA. ( K ) The serum IgG titers of normal individuals and PDAC patients to F-actin (sera diluted 1:100), to RUVBL2 (sera diluted 1:1000), and to HSPD1(sera diluted 1:1000), respectively, were measured by ELISA. The non-parametric t-test was used for comparison. Scale bars in B , F and I are 50um.

Article Snippet: Other antibodies and dyes used were: Phalloidin (Thermo Fisher, #A12379), MYH10 (Atlas Antibodies, #HPA047541), cytochalasin D (Thermo Fisher, #PHZ1063), ACTIN (Cell Signaling Technology, #3700S), RUVBL2 (Atlas Antibodies, #HPA067966), RUVBL1 (Atlas Antibodies, # HPA019947), HSPD1 (Atlas Antibody, #HPA050025), HSPA9 (Atlas Antibody, #HPA000898), COX4I1 (Atlas antibody, #HPA002485), KRT19 (Abcam, #ab203445), human IgG isotype controls (Biolegend, #403502 and #403702).

Techniques: Immunoprecipitation, SDS Page, Silver Staining, Mass Spectrometry, Staining, Dot Blot, Control, Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Western Blot, Comparison

Journal: bioRxiv

Article Title: Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies to self-antigens

doi: 10.1101/2023.03.20.533453

Figure Lengend Snippet: ( A ) 8-3 staining after siRNA knockdown of ACTB in MiaPaca2 cells, co-stained with phalloidin, MYH10 antibody and DAPI. ( B ) Measurement of 19-3 binding to recombinant RUVBL1 and RUBL2 proteins by ELISA. Positive controls using polyclonal rabbit antibodies anti-RUVBL1 (a-RUVBL1) or anti-RUVBL2 (a-RUVBL2). ( C ) 19-3 staining after siRNA knockdown of RUVBL2 in MiaPaca2 cells, co-stained with anti-RUVBL2 rabbit antibody and DAPI. ( D ) Co-staining of 15-7 with mitochondria markers COX4I1, HSPA9 and HSPD1 in MiaPaca2 cells. Scale bars in A, C and D are 50um.

Article Snippet: Other antibodies and dyes used were: Phalloidin (Thermo Fisher, #A12379), MYH10 (Atlas Antibodies, #HPA047541), cytochalasin D (Thermo Fisher, #PHZ1063), ACTIN (Cell Signaling Technology, #3700S), RUVBL2 (Atlas Antibodies, #HPA067966), RUVBL1 (Atlas Antibodies, # HPA019947), HSPD1 (Atlas Antibody, #HPA050025), HSPA9 (Atlas Antibody, #HPA000898), COX4I1 (Atlas antibody, #HPA002485), KRT19 (Abcam, #ab203445), human IgG isotype controls (Biolegend, #403502 and #403702).

Techniques: Staining, Knockdown, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies to self-antigens

doi: 10.1101/2023.03.20.533453

Figure Lengend Snippet: ( A ) Comparison of IgG titer to F-actin, RUVBL2 and HSPD1 of plasma samples diluted at 1/100 or 1/1000 by ELISA. Background signals from secondary antibody only are shown in a dashed line. ( B ) Comparison of IgG response to F-actin, RUVBL2 and HSPD1 between naïve treated (naive) and neoadjuvant treated (NAT) PDAC patients. The non-parametric t-test is used for comparisons.

Article Snippet: Other antibodies and dyes used were: Phalloidin (Thermo Fisher, #A12379), MYH10 (Atlas Antibodies, #HPA047541), cytochalasin D (Thermo Fisher, #PHZ1063), ACTIN (Cell Signaling Technology, #3700S), RUVBL2 (Atlas Antibodies, #HPA067966), RUVBL1 (Atlas Antibodies, # HPA019947), HSPD1 (Atlas Antibody, #HPA050025), HSPA9 (Atlas Antibody, #HPA000898), COX4I1 (Atlas antibody, #HPA002485), KRT19 (Abcam, #ab203445), human IgG isotype controls (Biolegend, #403502 and #403702).

Techniques: Comparison, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Nucleic Acids Research

Article Title: Cooperation of the NEIL3 and Fanconi anemia/BRCA pathways in interstrand crosslink repair

doi: 10.1093/nar/gkaa038

Figure Lengend Snippet: The RUVBL1/RUVBL2 complex is involved in psoralen-ICLs repair via the NEIL3 pathway. ( A ) 293T cells were transiently transfected with indicated plasmids, and co-IP were performed using anti-Flag antibody. WB of the inputs and IPs are shown to detect interactions. ( B ) 293T cells were transiently transfected with indicated plasmids, and co-IP were performed using normal IgG or anti-GFP antibody. WB of the inputs and IPs are shown to detect interactions. ( C ) NEIL3 protein levels were analyzed after knockdown of RUVBL1 or RUVBL2 in HeLa or U2OS cells. (D) RUVBL1 and RUVBL2 protein levels were analyzed by WB in WT and NEIL3 -/- cells. ( E, F ) Cell viability was measured and compared after knockdown of RUVBL1 or RUVBL2 and PUVA treatment in WT, NEIL3 -/- (E) and FANCA -/- (F) HeLa cells. CellTiter-Glo cell viability assays were performed.

Article Snippet: Membranes were blocked in 2% BSA in TBS-T for 1 h and then incubated overnight at 4°C with the following antibodies: anti-FLAG M2 antibody (Sigma-Aldrich #F1804), anti-HA-tag (Cell Signaling Technology #3724), anti-GFP (Cell Signaling Technology #2555), anti-beta-actin (Cell Signaling Technology #4967), anti-GAPDH (Cell Signaling Technology #2118), anti-NEIL3 (Proteintech, 11621-1-AP), anti-FANCA (Bethyl Laboratories #A301-980), anti-FANCD2 (Novus Biologicals #NB100-182), anti-FANCI (Santa Cruz Biotechnology #sc-271316), anti-γH2A.X (Ser139) (Cell Signaling Technology #2577), anti-PARP1 (Cell Signaling Technology #9542), anti-PARP3 (Novus Biologicals #NBP1-31415), anti-MCM7 (Cell Signaling Technology #3735), anti-RUVBL1 (Abcam #ab51500) and anti-RUVBL2 (BD Biosciences #612482).

Techniques: Transfection, Co-Immunoprecipitation Assay