Fig. 5

BTF3 interacts and stabilizes BMI1 protein (a) Kyoto Encyclopedia of Genes and Genomes (KEGG) terms of genes regulated by BTF3. (b-c) BMI1 protein levels were determined after incubation with cyclohexamide (CHX, 10 μg/mL) for the indicated time periods in PC3 Scr/shBTF3 (b) or 22RV1 Vec/BTF3 (c) cells. Left panel: Representative images of western blot assay. Right panel: the densitometric quantification of BMI1 normalized to GAPDH was plotted against various time points to determine its half life. BMI1(s): BMI1 with short exposure; BMI1(l): BMI1 with long time exposure. (d) PC3 and VCaP cells with Negative control (Scr) or BTF3 knockdown (shBTF3) were treated with control or 10 mM MG132 for 6 h and subjected to western blot analysis. (e) Effects of BTF3 on BMI1 ubiquitination. PC3 Scr/shBTF3 and 22RV1 Vec/BTF3 cells were incubated with MG132 (20 μM) for 6 h. Cell lysates were immunoprecipitated with BMI1 antibody and then immunoblotted with ubiquitin antibody. BTF3 and BMI1 in whole cell lysates (input) were shown. (f) Expression analysis of BMI1 with mutant β-TrCP binding site (mtBMI1) by western blot assay. (g) Identification of interacting proteins with BMI1 by co-IP and mass spectrometry. PC3 Scr/shBTF3 cells were incubated with MG132 (20 μM) for 6 h. Cell lysates were immunoprecipitated with BMI1 antibody and subjected to SDS–PAGE. (h) Possible proteins involved in regulation of BMI1 by BTF3 according to mass spectrometry. (i) Western blot analysis of GST-pull down assays. Whole-cell lysates from HEK293T cells were incubated with GST or GST-BTF3 fusion proteins coupled sepharose beads. After washing to remove unbound proteins, they were subjected to SDS–PAGE and western blot with antibodies against GST. (j-k) Co-immunoprecipitation assays of BTF3 and BMI1 in PC3 (g) and 293 T (h) cells. 293 T cells were transfected with plasmid for BMI1 and BTF3 expression. Cell lysates were immunoprecipitated with IgG control antibody or BTF3 antibody and then immunoblotted with BMI1 antibody. (l) Co-immunoprecipitation assays of BTF3 and truncate BMI1 in 293 T cells. 293 T cells were transfected with WT-BMI1, N-BMI1-FLAG or C-BMI1-FLAG plasmids and then subjected to co-immunoprecipitation assays with BTF3 antibody and then immunoblotted with FLAG antibody. * P < 0.05, **P < 0.01, ***P < 0.001.