Polyclonal anti actin antibody

K562-WT1-siRNA-GFP⁺ cells and K562-C-siRNA-GFP⁺ cells were lysed by the CelLytic M reagent (Sigma) for 15 minutes. Protein lysate was centrifuged at 12,000 rpm for 15 minutes, and the protein concentrations were determined using the Bradford protein assay (Sigma). Twenty micrograms of protein was separated on SDS-PAGE and then transferred to a PVDF membrane (Whatman) using blotting buffer for 1 hour. Then, the membrane was blocked for 1 hour and probed with specific primary antibodies (1 : 100 of polyclonal anti-WT1 antibody (Santa Cruz, C19) or 1 : 1000 of polyclonal anti-actin antibody (Santa Cruz, H196) or 1 : 1000 of anti-caspase-7 antibody (Sigma, C7724) in 1% skim milk at room temperature for 2 hours. Consequently, the membrane was detected with the immunocomplexes using horseradish peroxidase conjugated with either an appropriated dilution of goat anti-mouse or goat anti-rabbit secondary antibodies (Santa Cruz). The immunocomplex was detected with SuperSignal Pico Chemiluminescent Substrate for 5 minutes followed by film exposure.

24 h after transfection, COS-7 cells were gently washed twice with PBS, 50 × 10⁶ cells per 1 mL of lysis buffer (20 mM Tris/HCl (pH 8.2), 100 mM NaCl, 10 mM EDTA, 1% n-decyl-β-D-maltopyranoside (Anatrace, Maumee, OH, USA), 50 mMNaF, 1 mM orthovanadate, protease inhibitor mixture (Serva, Heidelberg, DE, Germany), 40 mM iodoacetamide (Sigma Aldrich, St. Louis, MO, USA), and reducing samples containing 100 mM dithiothreitol (Serva, Heidelberg, DE, Germany) were scraped off, and incubated on ice for 30 min. The insoluble material was removed by centrifugation at 3000 rcf for 3 min at 4°C. 20 µg of the total protein was separated on SDS-PAGE using a 10% polyacrylamide gel and were transferred onto PVDF membrane (Pall Corporation, Port Washington, NYn, USA) using semi-dry blotting apparatus. The transfer was run at 0.80 mA/cm2 for up to 1.5 h. Proteins were detected with polyclonal anti-GFP antibody (Exbio, Vestec, CZ, Czech Republic) according to manufacturer’s recommendations (1:2000 dilutions). For loading control, polyclonal anti-actin antibody (Santa Cruz, Dallas, TX, USA) was used. PVDF membranes were scanned using ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA).