Fig. 6

RBCK1 associates with YAP and modulates YAP stability in TNBC cells A The intracellular localization of RBCK1 and YAP was analyzed by immunofluorescence. MDA-MB-231 cells were cultured in normal medium before fixation. Intracellular localization of RBCK1 (red) and YAP (green) is shown. Nuclei (blue) were stained with 4’,6-diamidino-2-phenylindole (DAPI). B RBCK1 is mainly localized in the cytoplasm. YAP is mainly localized in the nuclear. We used a subcellular protein fractionation kit (Thermo Scientific, 78,840) for cytoplasm and nuclear separation experiment. C–D Co-IP experiment revealed the association between endogenous RBCK1 and YAP protein in MDA-MB-231 cells. MDA-MB-231 cells were collected with RIPA lysis buffer. CO-IP was performed using an antibody as indicated E–F RBCK1 full-length and deletion mutants are used in the study (Full-length, ΔUBL, ΔNZF, ΔRBR domains). YAP full-length and deletion mutants are used (Full length, ΔTA, ΔWW + ΔTA, ΔTBD, ΔTBD + ΔWW). G RBCK1 needs RBR domain to interact with YAP. HEK293 cells were transfected with 2 µg Myc-YAP and Flag-RBCK1 full-length or mutants (ΔUBL, ΔNZF, ΔRBR domains). 24 h later, the cells were collected with NP-40 lysis buffer. CO-IP was performed using an anti-Myc antibody. The interacting RBCK1 domains were detected by an anti-Flag antibody. H YAP needs TA domain to interact with RBCK1. HEK293 cells were transfected with 2 µg Flag-RBCK1 and Myc-YAP full-length or mutants (ΔTA, ΔWW + ΔTA, ΔTBD, ΔTBD + ΔWW). 24 h later, cells were harvested with NP-40 lysis buffer. The use of antibodies is shown in the figure. I–J RBCK1 did not further increase the level of YAP protein in TNBC cells in the presence of proteasome inhibitor MG132. MDA-MB-231 and BT549 cells were transfected with siControl or siRBCK1. 24 h later, the cells were treated with MG132 or vehicle for 6 h. The cell lysates were collected for western blot analysis. K The degradation effect of RBCK1 on YAP did not increase the levels of YAP protein any more in TNBC cell in the presence of the proteasome inhibitor MG132. HEK293 cells were transfected with 0.5 mg Flag-tag or Flag -RBCK1 plasmids. After 24 h, the cells were treated with 10 μM MG132/vehicle for 6 h. The cell lysates were prepared for western blot analysis. β-Actin was used as the internal reference. The results are representative of three independent experiments. L–M RBCK1 depletion increases the level of YAP half-life in TNBC cells. MDA-MB-231 and BT549 cells were transfected with siControl or siRBCK1. 24 h later, the cells were treated with 100 µM cycloheximide/vehicle for the specified time. The cell lysates were prepared for western blot analysis. The relative YAP density was measured by ImageJ software. β-Actin was used as the internal reference. The results are representative of three independent experiments. N Overexpression of RBCK1 reduced YAP half-life in HEK293 cells. HEK293 cells were transfected with 0.5 mg Flag-tag or Flag -RBCK1 plasmids. 24 h later, the cells were treated with 100 µM cycloheximide/vehicle for the specified times. The cell were collected for western blot analysis. The relative YAP density was measured by ImageJ software. β-Actin was used as the internal reference. The results are representative of three independent experiments