Fig. 2: LIMK2 negatively regulates SPOP’s levels by increased degradation.

a LIMK2 knockdown elevates SPOP protein level while LIMK2 overexpression reduces it. b Bar graph shows change in SPOP level with LIMK2 overexpression and knockdown. The data presented are mean ± SEM obtained from three experiments conducted independently. *P < 0.05 and ^#P < 0.05 compared to C4-2 control cells for LIMK2 and SPOP proteins, respectively. c 22Rv1 cells showing change in SPOP protein level with LIMK2 overexpression and knockdown. d Histogram shows change in SPOP protein level. The data are represented as mean ± SEM obtained from three experiments conducted independently. *P < 0.05 and ^#P < 0.05 compared to C4-2 control cells for LIMK2 and SPOP proteins, respectively. e, f rt-qPCR analysis depicting mRNA expression levels of SPOP in C4-2 cells infected with LIMK2 and LIMK2 shRNA retro/lentivirus. g, h rt-qPCR was used to quantify SPOP mRNA levels with LIMK2 overexpression and knockdown in 22Rv1 cells. i LIMK2 augments SPOP degradation in PCa cells. C4-2, LIMK2-C4-2 and LIMK2-CRISPR cells were treated with 10 μM cycloheximide for 3 and 6 h, and SPOP level was analysed. j Graphical representation of SPOP degradation rate in cells treated as in i. 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 0 h time point of each cell type. k Pattern of LIMK2 degradation in C4-2 and LIMK2-C4-2 cells at the time points where SPOP was chased using cycloheximide. l Quantification of the protein levels (relative to actin) as obtained from k (and two other independent experiments) upon normalisation with respect to the 0 h in each cell type. The data are presented as mean ± SEM acquired from three experiments independently. *P < 0.05, **P < 0.01, ***P < 0.001 vs 0 h time point of each cell type. m LIMK2 increases SPOP degradation by promoting its ubiquitylation. C4-2 cells were co-infected with 6x-His-ubiquitin (6x-His-Ub) along with LIMK2 for 30 h followed by MG132 treatment. Immunoprecipitated SPOP proteins were analysed for ubiquitylation using 6x-His antibody. The experiment was performed three times independently and representative data are included.