The expression of vinculin was used as a loading control. and its inhibitor. strain BL21 (DE3) (Stratagene, La Jolla, CA) and subsequently purified using the glutathione-Sepharose 4B batch method (GE Healthcare). translation was performed using the TnT? QuickCoupled transcription/translation systems (Promega, Madison, WI). Plasmid transfections were carried out using FuGENE HD transfection reagent (Roche Applied Science) according to the manufacturer’s instructions. When needed, after 24 h of transfection, cells were serum-starved for another 24 h before serum stimulation for the indicated times. Specific siRNAs targeting human PAK1 or control siRNAs were obtained from Cell Signaling Technology (Danvers, MA). The transfection of siRNA was performed twice at 24-h intervals with LY2452473 OligofectamineTM reagent (Invitrogen) according to the manufacturer’s protocol. 24 h after the second round of transfection, cells were serum-starved for 24 h before serum stimulation for the indicated times. LY2452473 Microarray Gene Expression Assays and Analysis Microarray gene expression assays and data analysis have been described previously (26). The datasets of differentially expressed genes between PAK1 WT and KO MEFs were analyzed using Ingenuity Pathway Analysis to discover relationships between the genes as well as their association with various processes and disorders.4 The Fisher’s exact test was used to identify significant functions and pathways represented within the respective datasets. Quantitative Real Time PCR (qPCR) Total RNA was isolated from cultured cells using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol, and 2 g of extracted RNA was converted to cDNA using the SuperScriptTM III First-strand Synthesis System for RT-PCR (Invitrogen). The resultant cDNA was subjected to qPCR by using the iQTM SYBR? Green Supermix (Bio-Rad) on an iCycler iQTM real time PCR detection system (Bio-Rad). The values for specific genes were normalized to human actin or mouse 18 S housekeeping controls. Mean values are Rapgef5 displayed S.D. The primers used were as follows: hACTIN_F, 5-TCC CTG GAG AAG AGC TAC GA-3, and hACTIN_R, 5-GTA CTT GCG CTC AGG AGG AG-3; hTF_F, 5-GCC LY2452473 CGG TAG AGT GTA TGG GCC AG-3, and hTF_R, 5-GCT CTG CCC CAC TCC TGC CTT-3; hTFPI_F, 5-AGT ATG GTG GAT GCC TGG GCA ATA-3, and hTFPI_R, 5-ACC TGG AAA CCA TTC GGA CCA TCT-3; m18 S_F, 5-CCG GAG CTA GGA ATA ATG GA-3, and m18 S_R, 5-CCC TCT TAA TCA TGG CCT CA-3; mTF_F, 5-AGA ACA CCC CGT CGC GCT TG-3, and mTF_R, GCT CTC CGC AAC AGT GCC GT-3; mTFPI_F, 5-TGT TGC LY2452473 TTA GCC TTG TTC CCG AGT-3, and mTFPI_R, TGC TTT GCA TGG ACC ATC ATC TGC-3. All qPCR primers were synthesized in Sigma. Western Blot and Immunoprecipitation Protein extracts were prepared by lysing the cells in RIPA buffer containing 50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 protease inhibitor mixture (Roche Applied Science), and 1 phosphatase inhibitor mixture I and II (Sigma), and protein concentrations were determined using Bio-Rad DC protein assay LY2452473 reagents (Bio-Rad). Cell extracts were then resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with the indicated antibodies. Detections were performed using the ECL reagents. For immunoprecipitation analysis, a total of 1 1 mg of protein material was incubated with 1 g of primary antibody overnight at 4 C on a rocket platform, followed by incubation with total 50 l of protein A/G PLUS-agarose (Santa Cruz Biotechnology) or Trueblot.
The expression of vinculin was used as a loading control