Fig. 3: LIMK2 is degraded by SPOP.

a SPOP overexpression decreases LIMK2 levels in C4-2 cells. b Bar graph shows change in LIMK2 level with SPOP overexpression. The data presented as mean ± SEM acquired from three experiments independently. **P < 0.01 vs the level of respective protein in C4-2 control cells. c SPOP ablation increases LIMK2 expression in C4-2 cells. Cells were infected with scrambled shRNA and SPOP-shRNA, and LIMK2 and SPOP levels were analysed. d Bar graph shows change in LIMK2 level with SPOP knockdown. *P < 0.05 and ^#P < 0.05 compared to C4-2 control cells for SPOP and LIMK2 proteins, respectively. e SPOP overexpression decreases LIMK2 levels in 22Rv1 cells. f Histogram shows change in LIMK2 protein level with SPOP overexpression. The data are represented as mean ± SEM of three experiments. *P < 0.05 and ^#P < 0.05 compared to C4-2 control cells for SPOP and LIMK2 proteins, respectively. g SPOP depletion increases LIMK2 levels in 22Rv1 cells. h Histogram shows change in LIMK2 protein level with SPOP knockdown. The data are presented as mean ± SEM obtained from three experiments independently. *P < 0.05 and ^#P < 0.05 compared to C4-2 control cells for SPOP and LIMK2 proteins, respectively. i rt-qPCR analysis representing mRNA levels of LIMK2 in C4-2 cells infected with SPOP retrovirus. j rt-qPCR analysis representing mRNA levels of LIMK2 in C4-2 cells infected with SPOP shRNA. k rt-qPCR was used to quantify LIMK2 mRNA level upon SPOP overexpression in 22Rv1 cells. l qPCR was used to quantify LIMK2 mRNA level upon SPOP knockdown in 22Rv1 cells. m SPOP increases LIMK2 degradation. C4-2, SPOP-C4-2 and SPOP shRNA-C4-2 cells were treated with cycloheximide (10 μM) for 2 and 4 h, and LIMK2 level was analysed. n Graphical depiction of LIMK2 stability. The results of densitometric scanning are presented graphically with LIMK2 signal normalised to tubulin signal. The data are presented as mean ± SEM acquired from three experiments independently. *P < 0.05 vs C4-2 control cells. o Pattern of SPOP degradation in C4-2, SPOP shRNA-C4-2 and SPOP-C4-2 cells measured at the times where LIMK2 was chased using cycloheximide. p Quantification of the protein levels (relative to actin levels) in o (and two other independent experiments) upon normalisation with respect to 0 h of each cell type. The data are presented as mean ± SEM acquired from three experiments independently. *P < 0.05, **P < 0.01 vs 0 h time point of each cell type. q SPOP degrades LIMK2 by enhancing its ubiquitylation. C4-2 cells were co-infected with 6x-His-ubiquitin and SPOP retrovirus for 30 h followed by 12 h MG132 treatment. LIMK2 was isolated and ubiquitylation analysed using 6x-His antibody. Each experiment was performed  for a minimum of three times independently and representative data are shown. r Immunofluorescence analysis to establish the location of LIMK2 in scrambled or SPOP shRNA treated C4-2 cells. s Subcellular localisation of LIMK2 in scrambled or SPOP shRNA-treated C4-2 cells as determined by subcellular fractionation. t Immunofluorescence analysis to establish the location of LIMK2 in scrambled or SPOP shRNA-treated 22Rv1cells.