Fig. 2: PRMT5 and UPS7 stabilizes G3BP2 by deubiquitination pathway.
From: USP7- and PRMT5-dependent G3BP2 stabilization drives de novo lipogenesis and tumorigenesis of HNSC
A, B Western blot and qPCR analysis of G3BP2 and PRMT5 expression in Tu686 and Tu212 cells transfected with siNC or siPRMT5. ns, no significant difference, **P < 0.01. C Tu686 cells were transfected with PRMT5 siRNAs and then incubated with or without MG132 (40 μM) for 6 h. Cell lysates were analyzed by immunoblotting. D Tu686 cells were transfected with negative control or PRMT5 siRNA and then applied with Cycloheximide (CHX, 50 μg/ml) for 2, 4 or 6 h. Immunoblotting analysis was used to measure the expression of G3BP2. *P < 0.05. E Cell lysates were immunoprecipitated with Flag-tag antibody and then immunoblotted by HA-tag antibody. F Cell lysates were immunoprecipitated with G3BP2 antibody before immunoblotting with HA-tag antibody. G HEK293 cells were transfected different deubiquitinating enzymes (DUBs) and then lysed for immunoblotting to detect the expression of G3BP2. H HEK293 cells were co-transfected with the indicated vectors for 48 h, followed by treated with MG132 for 6 h. Cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotting with anti-Myc antibody. I, J Western blot and RT-PCR analysis of USP7 expression in Tu212 and Tu686 cells transfected with siNC or siUSP7. ns, no significant difference. K Tu686 cells were transfected with negative control or USP7 siRNA and then applied with 50 μg/ml CHX for the indicated times and cell lysates were assessed by immunoblotting. *P < 0.05. L Western blot analysis of Tu686 and Tu212 cells with or without P5091, followed by treatment with DMSO and MG132 for 6 h, respectively. M Tu212 cells were transfected with G3BP2 and HA-Ub plasmids for 48 h, the purified G3BP2-Ubn was added 40 ng or 80 ng recombinant GST-USP7 proteins before immunoblotting analysis. N USP7 knockdown in Tu686 cells increased G3BP2 ubiquitination. Tu686 cells were co-transfected with HA-Ub and USP7 siRNA or control siRNAs, followed by treated with MG132 for 6 h. Cell lysates were immunoprecipitated using an anti-G3BP2 antibody and then subjected to immunoblotting.
