Mlc 2 m4401

For western blotting, cells transiently transfected with siRNA and synchronized in specific cell cycle stages were centrifuged at 1000g for 3 min. The pellet was washed with PBS and then lysed using RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The lysates were clarified by centrifugation at 8000g for 4 min at 4°C, diluted 1:1 with 2x Laemmli buffer (Sigma-Aldrich), incubated for 5 min at 95°C and loaded onto NuPage 4-12% Bis-Tris gradient gels (Invitrogen/Life Technologies). Primary antibodies used were 1:500 Anti-mDia1 (Cat. No. 96784 Abcam), 1:1000 Anti-CAPZB (AB6017 Millipore), 1:1000 Anti-CFL1 (3318s Cell signaling), 1:100,000 Anti-GAPDH (1D4, NB300-221 Novus Biologicals). Myosin amount and activity were assessed by running whole cell lysates on NuPage 14% Tris-Glycine gels (Invitrogen/Life Technologies). Primary antibodies used were 1:1000 pMLC-2 (Ser19) (3671S Cell Signaling), 1:2000 MLC-2 (M4401 Sigma-Aldrich), 1:2,000 Anti-GAPDH (1D4, NB300-221 Novus Biologicals), 1:2000 Anti-β-Actin (C4, sc-47778 Santa Cruz), and 1:2,000 anti-α-Tubulin (T5168 Sigma Aldrich). All secondary antibodies (Anti-rabbit IgG: NA934V; Anti-mouse IgG: NXA931 from GE Healthcare) were used at 1:5,000 for 1 hour at RT.