Figure 2: BAP1 stabilizes and deubiquitinates KLF5.

(a) BAP1 antagonized E3 ligase-mediated degradation of KLF5. HEK293FT cells were transfected with the BAP1 or KLF5 E3 ligases (WWP1, Fbw7γ and Smurf2) for 48 h. The cell lysates were analysed by WB. (b) BAP1 knockdown enhanced KLF5 protein degradation. MCF10A cells were transfected with Luc or varying BAP1 siRNAs. After treating the cells with cycloheximide (CHX) for an indicated time, the expression of endogenous KLF5 protein was analysed by WB (left panel). The band intensity of KLF5 for each time point was quantified by ImageJ and plotted (right panel). Experiments were repeated for three times, and a representative experiment is presented. Error bars represent s.d. Every experimental group was compared with the control Lucsi group, *P<0.05, t-test. (c) BAP1 overexpression stabilized the KLF5 protein. KLF5 was co-expressed with BAP1 or BAP1-C91S in HEK293FT cells. After the cells were treated with CHX, KLF5 protein levels were analysed by WB (left panel). GST was used as transfection and loading controls. Quantitative data are shown in the right panel. Experiments were repeated for three times, and a representative experiment is presented. Error bars represent s.d. Every experimental group was compared with the vector group, *P<0.05; **P<0.01, t-test. (d) BAP1 decreased KLF5 ubiquitination in HEK293FT cells. KLF5-3 × Flag and HA-Ub were co-expressed with BAP1, BAP1-C91S or A20. After the cells were treated with MG132 for 6 h, KLF5 proteins were immunoprecipitated and the polyubiquitinated KLF5 proteins were detected by WB using an anti-HA antibody. (e) BAP1 deubiquitinates KLF5 in vitro. Ubiquitinated KLF5 was purified from MG132-treated HEK293FT cells and then incubated with purified GST-tagged BAP1 or BAP1-C91S in vitro. The polyubiquitinated KLF5 proteins were examined by WB. (f) Stable BAP1 knockdown HCC1806 cells and Lucsh cells were treated with MG132 (20 μM) for 6 h. Cell lysates were immunoprecipitated with the anti-KLF5 antibody and KLF5 ubiquitination was examined by WB using the anti-Ub antibody. (g) The HA-Ub plasmid was transiently transfected into Lucsh and BAP1sh HCC1806 cells for 48 h. The cells were treated with MG132 (20 μM) for 6 h. Extracts were immunoprecipitated with KLF5 antibody, and KLF5 ubiquitination was examined by WB using the anti-Ub antibody.