Fig. 1: G3BP1 interacts with SPOP and acts as a negative regulator of SPOP ubiquitin ligase.

a 22Rv1 cell lysates were subjected to immunoprecipitation (IP) with IgG and anti-G3BP1 antibody and immunoblotted with anti-SPOP and anti-G3BP1 antibodies. n = 3. b HEK 293T cells were transiently transfected with His-Ub and the indicated construct(s) for 48 h. Cell lysates were subjected to His-Ub pulldown using nickel-NTA beads under denaturing conditions, followed by SDS–PAGE, and immunoblotting with the anti-FLAG antibody. Expression of FLAG-AR, MYC-G3BP1, and HA-SPOP was detected by immunoblotting. α-tubulin served as an internal loading control. n = 3. c DEK ubiquitination (Ub) was reconstituted in vitro using affinity-purified recombinant proteins as indicated. Purified proteins and ubiquitinated DEK were subjected to immunoblotting with the indicated antibodies. All protein concentrations were in nM except Ub, which was in µM. n = 3. d Immunoblotting of cytosolic and nuclear fraction of LNCaP-sgCtrl and LNCaP-sgG3BP1 cells with the indicated antibodies. Results from three independent experiments were quantitated by densitometry and relative protein expression of SRC3, AR, and TRIM24 in LNCaP-sgG3BP1 cells relative to those of LNCaP-sgCtrl were plotted. Error bars, ±S.E.M. Paired t-test. e Cycloheximide (CHX)-chase analysis to determine the half-life of endogenous TRIM24 in 22Rv1-sgCtrl and 22Rv1-sgG3BP1 cells. The percentage of TRIM24 remaining was graphed at the time points indicated. n = 3 biologically independent experiments. Error bars, ±S.E.M. Paired t-test. p value is indicated in figure. Source data are provided as a Source Data file. WCL whole cell lysate, Cyt cytoplasmic extract, Nu nuclear extract.