Anti cofilin

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.

Y27632, Lucifer yellow and anti-Cx43 antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-RhoA, anti-Rac1, anti-Cdc42, anti-RhoGDI, and anti-cofilin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-p190RhoGAP antibody was purchased from Millipore (Lake Placid, NY, USA). Anti-phospho-RhoA (Ser188), anti-phospho-LIMK1/2 (Thr508 and Thr505), anti-LIMK1, and anti-Cx43 antibodies were purchased from Abcam (Cambridge, MA, USA). The anti-phospho-cofilin antibody (Ser3) was obtained from Cell Signaling Technology (Danvers, MA, USA).

Bone marrow and FLC-derived CD45⁻ erythroblasts were lysed with protease inhibitors (Protease Inhibitor Cocktail Tablets, Roche), quantified, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10–12% polyacrylamide gels, and transferred to nitrocellulose membrane (Bio-Rad). Membranes were blocked for 1 h in TBST (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.05% Tween 20) containing 5% skim milk or 5% BSA, followed by overnight incubation with commercially available primary antibodies (anti-plek2 1:1000 (Proteintech, 11685-1-AP); anti-cofilin 1:1000 (Santa Cruz, sc-376476); anti-Rac1 1:1000 (Cell Signaling Technology, 2465); anti-VDAC1 1:1000 (Millipore, MABN504); anti-cytochrome c 1:1000 (Abcam, ab90529); and anti-GAPDH 1:4000 (Invitrogen)). Blots were washed and incubated for 1 h at room temperature (RT) with the secondary antibody (horseradish peroxidase (HRP) conjugated (Thermo Scientific)). Immunoreactive bands were visualized by the enhanced chemiluminescence (ECL) method (Amersham Bioscience) according to standard procedures. For mitochondria fractionation and activated Rac1 pull-down (Thermo Scientific, 89,874 and 16,118, respectively), samples were processed according to the manufacturer’s instructions.