Fig. 2: Molecular determinants of crosstalk between G3BP1 and SPOP.
From: G3BP1 inhibits Cul3SPOP to amplify AR signaling and promote prostate cancer
a, b Schematic illustration of a series of SPOP deletion mutants and G3BP1 deletion mutants used in this study. “Inverted triangle” shows SPOP dimerization sites and mutations (L186D, L190D, L193D, I217K). c, d Immunoprecipitation and immunoblotting of WCL derived from HEK 293T cells transfected with the indicated full length (FL) or (c) SPOP or (d) G3BP1 deletion constructs. β-actin served as the loading control. HA-MATH: HA-tagged MATH domain of SPOP; HA-BTBMut: HA-tagged BTB domain of SPOP with mutated dimerization sites; N: NTF2 (1-138 aa), M1: G3BP1 (139-466 aa), M4: G3BP1 (222-466 aa), M2: G3BP1 (139-338 aa), M3: G3BP1 (222-338 aa), C: RRM (338-466 aa). n = 3. e Immunoprecipitation and immunoblotting of WCL derived from 22Rv1 cells transfected with the indicated construct(s). n = 3. f PLA assay for the FLAG–G3BP1 and its deletion mutants with endogenous SPOP. Each red dot represents an interaction (scale bar, 10 μm). Representative immunofluorescence images of 22Rv1 cells transfected with FLAG-G3BP1, FLAG-NTF2, FLAG-RRM, or FLAG-PxxP. A graph showing percentage of cells with G3BP1 and SPOP interaction. n = 3 biologically independent experiments. Error bars, ±S.E.M. g Parental 22Rv1 cells were transfected with either HA-SPOPWT or HA-SPOPF133V or HA-SPOPF102C. After 48 h, cell lysates were immunoprecipitated with anti-G3BP1 antibody and immunoblotted with either anti-HA-SPOP or anti-G3BP1 antibody. NT no transfection. n = 3. h HEK 293T cells were transiently transfected with the indicated construct(s) for 48 h. Ni-NTA pull-down products or WCL were blotted for the indicated proteins. α-tubulin serves as the loading control. n = 3. Source data are provided as a Source Data file. WCL whole cell lysate.
