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. 2019 Aug 1;9(8):1583–1606.

Figure 7.

Figure 7

RILPL2 inhibits BC cell proliferation and migration by down-regulating TUBB3 stability. (A) Exogenous interaction between RILPL2 and TUBB3 in 293 cells by co-immunoprecipitation. (B) RILPL2-shRNA or scr-shRNA were transfected into RILPL2-overexpressing T-47D and MDA-MB-231 cells. TUBB3 expression was examined by western blotting (n = 3). (C) Level of TUBB3 mRNA was examined by qPCR in RILPL2-overexpressing T-47D and MDA-MB-231 cells. (D) RILPL2 mediated TUBB3 stabilization in BC cells. RILPL2-expressing MDA-MB-231 cells were treated with CHX (50 μM) for the indicated times and analyzed for endogenous TUBB3 by western blotting. (E) RILPL2 overexpression increased TUBB3 ubiquitination in breast cancer cells. HA-Ubiquitin was electroporated into Vector- or RILPL2-overexpressing MDA-MB-231 cells. Cells were treated with MG132 (20 μM) for 2 h. TUBB3 complex in resulting lysates was examined using an antibody against Ubiquitin (Ub). (F-H) Vector or TUBB3 were transfected into RILPL2-expressing T-47D and MDA-MB-231 cells. Cell growth rate (F) monitored by MTT assay in T-47D and MDA-MB-231 cells. Cell wound (G) and invasiveness (H) were monitored in MDA-MB-231 cells (n = 3). Results are presented as means ± SD. The statistical significance was assessed by Student’s t-test; *P < 0.05, **P < 0.01.