Fig. 1: LIMK2 directly phosphorylates SPOP and regulates its nuclear residence.

a LIMK2 directly phosphorylates WT-SPOP protein in vitro. The first lane is LIMK2 alone, the middle lane is SPOP with LIMK2 and the third lane is SPOP alone. The kinase reaction was carried out in 25 µl volume containing 1× kinase buffer and 1 µCi [γ-³²P] ATP. b LIMK2 does not interact with SPOP in CRPC cells. C4-2 and 22Rv1 cell lysates were used for immunoprecipitation with IgG antibody or LIMK2 antibody, followed by immunoblotting with SPOP antibody. c SPOP and LIMK2 do not interact with each other. PCa cell lysates were used for immunoprecipitation with IgG or SPOP antibody, followed by immunoblotting with LIMK2 antibody. d Immunofluorescence analysis to detect SPOP localisation in response to LIMK2 inhibitor in C4-2 cells. e Immunofluorescence analysis to establish the location of SPOP in scrambled or LIMK2 shRNA-treated C4-2 cells. f Subcellular localisation of SPOP in scrambled or LIMK2 shRNA-treated C4-2 cells as determined using cellular fractionation. g Immunofluorescence analysis to detect SPOP localisation in response to LIMK2 inhibitor in 22Rv1 cells. h Immunofluorescence analysis to establish the location of SPOP in scrambled or LIMK2 shRNA-treated 22Rv1 cells.