rabbit  (Cell Signaling Technology Inc)

Journal: PLOS Biology

Article Title: CUL5 E3 ubiquitin ligase regulates the evasion of bladder cancer cells to CD8 + T cell-mediated killing by inhibiting autophagy

doi: 10.1371/journal.pbio.3003647

Figure Lengend Snippet: (A) The expression levels of LC3B and P62 in T24 cells transfected with vector, Flag-Rubicon-L, or Flag-Rubicon-S were detected by western blotting. (B) T24 cells were transfected with vector, Flag-Rubicon-L or Flag-Rubicon-S plasmids, and those co-transfected with mCherry-EGFP-LC3B. The autophagosomes with yellow puncta and autolysosomes with red puncta. Bar: 10 μm. (C) T24 cells were transfected with the vector, Flag-Rubicon-L, and Flag-Rubicon-S plasmids, and then co-cultured with CD8 + T cells, and cell viability was measured by CCK-8. (D) Western blotting with the indicated antibodies in T24 cells transfected with scramble, sh-RUBCN-L or sh-RUBCN-S. The efficiency of RUBCN-L knockdown and RUBCN-S knockdown in T24 cells was detected by agarose gel electrophoresis (left) and qRT-PCR (right). (E) Co-IP assay using antibody specific for Rubicon showed that Rubicon interacted with UVRAG and Beclin1 in T24 cells. The precipitate was subjected to western blotting with the antibodies against Rubicon, UVRAG, and Beclin1. (F) Co-IP assay using antibody specific for Flag showed that Flag-Rubicon-S interacted with UVRAG and Beclin1(Right), while Flag-Rubicon-L could not bind to UVRAG and Beclin1 in T24 cells (Left). The precipitate was subjected to western blotting with the antibodies against Flag, UVRAG, and Beclin1. (G) The expression levels of LC3B and P62 in CUL5-KO T24 cells were detected by western blotting. (H) CUL5-WT, CUL5-KO#1, and CUL5-KO#2 T24 cells were transfected with the mCherry-EGFP-LC3B. The autophagosomes with yellow puncta and autolysosomes with red puncta. Bar: 10 μm. (I) Western blotting with the indicated antibodies in CUL5-WT and CUL5-KO T24 cells transfected with scramble or sh-PTBP1#1. (J) Western blotting with the indicated antibodies in CUL5-WT and CUL5-KO T24 cells transfected with scramble or sh-RUBCN-L, and agarose gel electrophoresis for analysis of RUBCN isoforms. (K) CUL5-WT, CUL5-KO#1, and CUL5-KO#2 T24 cells were transfected with the scramble or sh-RUBCN-L, and then co-cultured with CD8 + T cells, and cell viability was measured by CCK-8. (L) Western blotting with the indicated antibodies in CUL5-WT and CUL5-KO T24 cells transfected with scramble or sh-RUBCN-S, and agarose gel electrophoresis for analysis of RUBCN isoforms. (M) CUL5-WT, CUL5-KO#1, and CUL5-KO#2 T24 cells were transfected with the scramble or sh-RUBCN-S, and then co-cultured with CD8 + T cells, and cell viability was measured by CCK-8. (N) The expression levels of MHC-I (HLA-A, -B, -C) in CUL5-KO T24 cells co-cultured with CD8 + T cells were detected by western blotting. Data are presented as the means ± SD from three independent experiments. Student t test was applied to analyze and compare the data in C, D, K, and M. ns, nonsignificant; ** P < 0.01; *** P < 0.001. The raw data underlying all figures can be found in . Original blots and gels can be found in .

Article Snippet: Antibodies used included primary antibodies against CUL5 (Abclonal, A5369), β-Actin (Proteintech, 66009-1-Ig), PRMT5 (Proteintech, 18436-1-AP), THOC2 (Proteintech, 55178-1-AP), THRAP3 (Proteintech, 19744-1-AP), SNRNP200 (Proteintech, 23875-1-AP), PTBP1 (Proteintech, 12582-1-AP), SF3B1(Proteintech, 27684-1-AP), PRPF8(Proteintech, 11171-1-AP), SF3B2 (Proteintech, 10919-1-AP), HNRNPC (Proteintech, 11760-1-AP), LC3B (Abclonal, A5618), P62 (Proteintech, 18420-1-AP), Rubicon (Proteintech, 21444-1-AP), UVRAG (Proteintech, 29190-1-AP), Beclin1 (Proteintech, 11306-1-AP), HLA-class I (Proteintech, 15240-1-AP), Rabbit control IgG (Abclonal, AC005), Mouse control IgG (Abclonal, AC011), Mouse anti-HA tag (Abclonal, AE008), Rabbit anti-HA tag (Abclonal, AE036), Mouse anti-Flag tag (Abclonal, AE005), and Rabbit anti-Flag tag (Abclonal, AE004); HRP-conjugated secondary goat anti-mouse (Proteintech, SA00001-1), or goat anti-rabbit (Proteintech, SA00001-2) antibodies.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Cell Culture, CCK-8 Assay, Knockdown, Agarose Gel Electrophoresis, Quantitative RT-PCR, Co-Immunoprecipitation Assay