Cas 1177865 17 6

Bovine serum albumin (BSA) A7906, progesterone, Coomassie brilliant blue-G, sennoside A, TRITC-labeled phalloidin, fluorescein isothiocyanate (FITC)-labeled PNA, phosphatase inhibitor cocktail P5726, and protease inhibitor cocktail P8340 were purchased from Sigma-Aldrich. Eosin Y was purchased from Biopack. Membrane-permeable Exo enzyme C3 Transferase (C4) was obtained from Cytoskeleton. CAS 1177865-17-6 was obtained from Merck. Cyclosporin A and OA were obtained from Cayman Chemical. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and HRP anti-mouse IgG from Vector Laboratories. PF-3758309 from Medkoo Biosciences. Antibody anti-rabbit IgG-Alexa Fluor 568 from Invitrogen, Thermo Fisher Scientific; while anti-14-3-3 (pan 14-3-3 b8) from Santa Cruz Biotechnology. Anti-COFILIN, anti-phospho-SSH1 (pSSH1; Ser978), anti-phospho-LIMK1/2 (pLIMK1/2; Thr508), and anti-phospho-COFILIN (pCOFILIN; Ser3) antibodies were purchased from Cell Signaling. Anti-PAK4 from Proteintech. Anti-β-tubulin E7 was obtained from Developmental Studies Hybridoma Bank University of Iowa. PF-3758309, cyclosporine A, OA, and sennoside A were dissolved in DMSO; C4, CAS 1177865-17-6, and Eosin Y were dissolved in hexa-distilled water, and phalloidin-TRITC and PNA-FITC were dissolved in PBS.

For treatment with wortmannin to assess Cdc42 activation, cells were treated for 3 h at 37 °C, followed by stimulation with insulin for the indicated times. Cells were lysed and analyzed for Cdc42 activation as described above. For analysis of cell pairs, cells were plated in Matrigel and then treated for 16 h in 200 nM wortmannin followed by fixation and labeling as indicated. For Rac1 inhibitor studies, cells were plated in 2% Matrigel containing 50 μm CAS 1177865–17–6 (EMD Millipore) and incubated for 16 h followed by fixation and labeling.