Assay for MLC phosphorylation.

In a separate set of experiments, permeabilized control and TNFα-exposed ASM strips were separately incubated (at 22°C) in pCa 9.0, 6.1, and 4.0 solutions to induce relaxation, 50% of F_max, and F_max, respectively. After 20 min of incubation, ASM strips were snap-frozen in liquid nitrogen. Frozen ASM strips were thawed at room temperature, minced with a microscissors, and homogenized in ice-cold radioimmunoprecipitation assay lysis buffer (RIPA, Cell Signaling Technology, Danvers, MA) supplemented with 1 mM PMSF, 1× phosphatase inhibitor (PhosSTOP Easypack Roche, Mannheim, Germany), and 1× protease inhibitor (cOmplete Mini, Roche). Lysed samples were centrifuged at 12,000 rpm for 20 min in a cold room, and supernatants were collected. Protein concentration of each sample was estimated by standard Bradford assay (Bio-Rad protein assay system, Bio-Rad, Hercules, CA) (7). Each sample was separated on a 15% SDS gel (20 µg per well) and transferred to a polyvinylidene difluoride (PVDF) membrane (paired samples from control and TNFα-exposed groups were run on the same gel). Transferred proteins were detected with a specific antibody for p-MLC [1:1,000 dilution, rabbit polyclonal antibody to MLC (phosphorylated at S20); Ab2480, Abcam, Cambridge, MA] (42), then membranes were stripped and probed again for total MLC (1:1,000 dilution, monoclonal anti-MLC antibody, clone MY-21; Sigma-Aldrich) (22). Total MLC and p-MLC bands were visualized using an enhanced chemiluminescence technique and quantified using a Kodak Image Station (Kodak, Los Angeles, CA). Data are presented as the ratio of p-MLC to total MLC for each set.