Pgex 5x 1 2

GFP-, DsRed-, Flag-, and GST-tagged
PAK4 were constructed by polymerase chain reaction (PCR) and sub-cloned into pEGFP-C1, DsRed, pcDNA3.1-Flag (Invitrogen), and pGEX-5X-1/2 (GE Healthcare, Bethesda, USA) vectors, respectively. The full length CORO1C was amplified by PCR using cDNA derived from SGC-7901 cells as a template and cloned into pEGFP-C1, DsRed, pcDNA3.1-Flag and pGEX-4T-2 vectors. CORO1C deletion constructs were obtained by PCR and cloned into pGEX-4T-2. The site-directed mutagenesis (PAK4S99A or PAK4S99D) was generated from PAK4 WT using the QuikChange kit (Stratagene, La Jolla, USA) according to the manufacturer′s instructions. eYFP-RCC2 was a kind gift from Prof. Sheng Xiao of Harvard Medical School (Boston, USA).

pcDNA-EGFP-PAK6 wild-type (WT)/K436A (KA, kinase-dead) were gifts from Dr. G. M. Bockoh (The Scripps Research Institute). Flag/GST-tagged PAK6 and PAK1,4,5 were constructed by polymerase chain reaction (PCR) and subcloned into pcDNA3.1-Flag (Invitrogen) and pGEX-5X-1/2 (GE Healthcare) vectors, respectively. Flag-tagged SIRT4 was purchase from GeneChem (Shanghai, China). HisA-ANT2 WT was constructed by PCR and subcloned into pcDNA3.1/Myc-HisA (Invitrogen) vectors, respectively. The site-directed mutagenesis (ANT2 T107A mutant, ANT2 K105R mutant and ANT2 T107A/K105R mutant) was generated from ANT2 WT using the QuikChange kit (Stratagene), according to the manufacturer's instructions. The myc-ubiquitin constructs were previously generated in our laboratory.