Anti actin c 2 antibody

We generated wt- or mutated-tax (tagged c-Myc) pcDNA3.1(-)-c-Myc vectors based on pcDNA3.1(-) (Invitrogen, Waltham, MA). 293 T cells (2 × 10⁶) were harvested 24 h after transfection with 6 μg of wt- or mutated-tax (tagged c-Myc) pcDNA3.1(-)-c-Myc vectors using 16 μl of HilyMax (Dojindo Laboratories). After cell lysis in 200 μl of RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1 mM EDTA) with protease inhibitor cocktail and phosphatase inhibitor cocktail, 30 μg of cell proteins were separated on the precast gel for SDS-PAGE (197-15011; FUJIFILM, Osaka, Japan). Separated proteins were blotted onto polyvinylidene difluoride membrane (5400412; ATTO, Tokyo, Japan). About 0.1 μg/ml anti-Myc antibody (M192-3; MBL, Tokyo, Japan) and 0.1 μg/ml anti-Actin (C-2) antibody (sc-8432; Santa Cruz Biotechnology, Dallas, TX) were used as primary antibodies staining. secondary reactions with HRP were performed using Pierce^TM Fast Western Blot Kit, ECL Substrate (35055; Thermo Fisher Scientific) according to the manufacturer’s instruction. Chemiluminescent detection was performed on a ChemiDoc^TM Touch Imaging system (BIO-RAD).