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Image Search Results
Journal: bioRxiv
Article Title: Epstein–Barr Virus BALF0/1 Subverts the Caveolin and ERAD Pathways to Target B-cell Receptor Complexes for Degradation
doi: 10.1101/2024.01.04.574276
Figure Lengend Snippet: BALF0/1 mediated BCR degradation requires the Ig heavy chain cytoplasmic tail (A) Immunoblot analysis of WCL from Cas9+ P3HR-1 cells expressing the indicated CD79A targeting sgRNA, 4-HT induced into lytic cycle for 24 h. (B) Immunoblot analysis of WCL from Cas9+ P3HR-1 cells expressing the indicated CD79B sgRNA, 4-HT induced into lytic cycle for 24 h. (C) Immunoblot analysis of WCL from Cas9+ P3HR-1 cells expressing the indicated CD79A and CD79B sgRNA, 4-HT induced into lytic cycle for 24 h. (D) Mean ±SEM percentage of cells with PM IgM signals as in Figure S6 of P3HR-1 cells expressing the indicated control or dual CD79A/CD79B sgRNA, uninduced or 4-HT induced, as indicated, using data from 12 randomly selected panels of 240 cells from n=3 replicates, analyzed using the ImageJ ComDet plugin. (E) Immunofluorescence analysis of CD79A, CD79B, IgM and calnexin in P3HR-1 cells expressing control sgRNA and 4-HT induced for lytic replication in the presence of bortezomib for 24h. (F) Fluorescence intensity line scanning for calnexin (magenta) and IgM (green) signals from the white rectangle region (E). (G) Immunofluorescence analysis of IgM, calnexin, CD79A and CD79B in Cas9+ P3HR-1 cells expressing CD79A and CD79B sgRNA. Cells were 4-HT induced for lytic replication and treated with bortezomib for 24 h. (H) Fluorescence intensity line scanning for calnexin (magenta) and IgM (green) signals from the white rectangle region in (G). (I) Mean ±SEM percentage of cells with overlapping calnexin and IgM signals as in panel E, G and Figure S6 of P3HR-1 cells expressing the indicated control or dual CD79A/CD79B sgRNA, uninduced or 4-HT induced in the absence or presence of bortezomib as indicated, using data from 12 randomly selected panels of 240 cells from n=3 replicates, analyzed using the ImageJ ComDet plugin. (J) Schematic model of the BCR complex immunoglobulin heavy (IgH) chain, light chain and the CD79A and CD79B signaling chains. The C’ IgM cytoplasmic tail residues are shown. (K) Immunoblot analysis of WCL from 293T cells transiently expressing wildtype (WT) or cytoplasmic tail deleted (ΔKVK) EGFP-tagged IgM heavy chain alone or together with either BXLF1 or BALF0/1. (L) Immunoblot analysis of WCL from 293T transiently expressing the IgA heavy chain, alone or together with BALF0/1 or BXLF1. Statistical analysis was performed with Student’s t-test unless otherwise specified. ns p > 0.05. White bars indicate scale. See also Figure S 6. Blots are representative of at least n=2 replicates.
Article Snippet: Antibodies used for immunoblot analysis in this study were: anti-Human IgM goat polyclonal antibody (Southern Biotech #2020-01), anti-Human IgG goat polyclonal antibody (Southern Biotech #2040-01), anti-HA-Tag (C29F4) rabbit mAb (Cell Signaling #3724), anti-GAPDH (D16H11) XP® rabbit mAb (Cell Signaling #5174),
Techniques: Western Blot, Expressing, Control, Immunofluorescence, Fluorescence