Hrp labeled anti mouse igg

SDS-PAGE was performed using standard techniques. Separated proteins were transferred to an Immobilon-P PVDF membrane (Millipore). The membrane was incubated with blocking buffer [5% skimmed milk in TBS-T (20 mM Tris, 150 mM NaCl, 0.05% Tween-20)] for 30 min. The membrane was then incubated with the primary antibodies in blocking buffer overnight at 4°C, followed by the secondary antibodies for 1 hour at 25°C. The primary antibodies were diluted as follows: mouse anti--tubulin monoclonal antibody (1:2,000) (B-5-1-2; SIGMA-Aldrich), mouse anti-acetyl--tubulin monoclonal antibody (1:500) (6-11B-1; SIGMA-Aldrich), mouse anti--actin monoclonal antibody (1:2,000), mouse anti-vinculin monoclonal antibody (1:500), mouse anti-GAPDH monoclonal antibody (1:30,000) (6C5; Millipore), rabbit anti-NMHC-IIA polyclonal antibody (1:60,000), and rabbit anti-NMHC-IIB polyclonal antibody (1:2,000). The secondary antibodies (Bio-Rad Laboratories, Hercules, CA, USA) were diluted as follows: HRP-labeled anti-mouse IgG (1:10,000) and HRP-labeled anti-rabbit IgG (1:10,000). The chemiluminescent signal was produced using the Immobilon Western Chemiluminescent HRP Substrate (Millipore) and detected using a LAS-3000 (Fujifilm, Tokyo, Japan). The percentages of cytoskeletal fractions were calculated by densitometry using ImageJ software.