Ab125189

4% paraformaldehyde was used to fix MDAMB231 cells for 15 min, 0.25% Triton X-100 to permeabilize them for 15 min, and 3% BSA to block them for 1 h at room temperature. The rabbit anti-RNF31 antibody (ab125189, Abcam, 1:400), mouse anti-YAP antibody (SC-101199, 1:200) and mouse anti-PD-L1 antibody (1:400, 66,248–1-Ig, Proteintech) were used. The Alexa Flour 647 (Invitrogen) anti-mouse or Alexa Flour 488 anti-rabbit secondary antibodies (Invitrogen). The nuclei were stained with DAPI (Beyotime). Samples with only secondary antibodies and no primary antibodies were used as negative controls. Images were captured with a Nikon A+ laser scanning confocal system.

Cells were lysed with Western and IP lysis buffer (P0013J, Beyotime) with a protease inhibitor cocktail (Roche). Total cell lysates were incubated with 30 μl of Protein Agarose (Beyotime, P2012) and IgG (Beyotime, 1:50) for preclearing 2 h at 4 °C, and immunoprecipitation was then performed with an indicated antibody for 4 h at 4 °C. As a negative control, Rabbit IgG (Beyotime, A7016, 1:50) or Moues IgG (Beyotime, A7028, 1:50) was used. Separation of proteins by SDS-polyacrylamide gel electrophoresis (PAGE) and transfer to nitrocellulose membranes (Millipore) were carried out. The antibodies were used as follows: anti-RNF31 (ab125189, Abcam, 1:2000); anti-HA (MMS-101R, COVANCE, 1:2000); anti-Flag (20543–1-AP, Proteintech, 1:2000); anti-YAP (14,074, Cell Signaling Technology, 1:2000; SC-101199, Santa Cruz, 1;500); PD-L1 (13,684, Cell Signaling Technology; 66,248–1-Ig, Proteintech); anti-Tubulin (11224–1-AP, Proteintech, 1:5000); anti-Histone-H3 (17168–1-AP, Proteintech, 1:1000) anti-Myc (Ab9106, Abcam, 1:2000); and anti-β-actin (8H10D10, Cell Signaling Technology, 1:10000). AffiniPure goat anti-mouse peroxidase-conjugated antibody IgG-HRP (A0216, Beyotime, 1:5000) or goat anti-Rabbit IgG-HRP (A0208, Beyotime, 1；5000) secondary antibodies was incubated with the membranes after three washes with PBST. The signals were detected with the ECL Kit (Meilunbio, #MA0186).

The streptavidin-peroxidase-biotin (SP) immunohistochemical method was used to measure RNF31 and YAP protein expression in 52 TNBC breast tissues. For primary antibodies, sections of tissues were incubated with RNF31 (ab125189, Abcam, 1:200) or YAP (14,074, Cell Signaling Technology, 1:200) antibodies overnight at 4 °C, while sections of xenografts from BALB/c mice with rat monoclonal anti-CD8 (eBioscience, 1:200). Immunoreactivity was measured as depicted in previous paper [25 (link)]. This usage of clinical samples was reviewed and approved by the Ethical Board at Shandong University with written informed consent from all the patients.