A, B: HeLa cells were transfected with control shRNA (pSuper-scr-shRNA) or shRNA specifically-targeting PKD3 (pSuper-PKD3-shRNA) and next day also transfected with FLAG-tagged PAK4 (A) or LIMK1 (B), as indicated. 48 hours after initial infection, cells were lysed and PAK4 (A) or LIMK1 (B) was immunoprecipitated (anti-FLAG). Samples were subjected to SDS-PAGE, transferred to nitrocellulose and immunostained for PAK4 activity (anti-pS474-PAK4) (A) or LIMK1 activity (anti-pT508-LIMK1) (B). After stripping samples were re-probed with anti-FLAG for total PAK4 (A), or total LIMK1 (B). PKD3 knockdown was controlled by Western blotting (anti-PKD3) and equal loading was controlled by Western blotting for β-actin (anti-β-actin). C: HeLa cells were transfected with control shRNA (pSuper-scr-shRNA) or shRNA specifically-targeting PKD3 (pSuper-PKD3-shRNA) and next day also transfected with FLAG-tagged cofilin, as indicated. 48 hours after initial infection, cells were lysed, samples subjected to SDS-PAGE, transferred to nitrocellulose and immunostained for pS3-phosphorylated cofilin (anti-pS3-cofilin), cofilin (anti-FLAG), PKD3 knockdown (anti-PKD3) or β-actin (anti-β-actin) as loading control. D: HeLa cells were transfected with control shRNA (pSuper-scr-shRNA) or shRNA specifically-targeting PKD3 (pSuper-PKD3-shRNA) and next day also transfected with FLAG-tagged constitutively-active PAK4 (PAK4.CA), as indicated. 48 hours after initial infection, cells were lysed, samples subjected to SDS-PAGE, transferred to nitrocellulose and immunostained for pS3-phosphorylated cofilin (anti-pS3-cofilin), cofilin (anti-cofilin), PKD3 knockdown (anti-PKD3) or PAK4.CA (anti-FLAG). Experiments shown in A-D were performed at least three times with similar results.