Anti mlc2

Then, 15 h post transfection, cells were treated with either 1 µM PMA (a PKC agonist, Sigma-Aldrich, St Louis, MO, USA) or 0.1% DMSO for 3 h, then washed in PBS, and lysed in RIPA buffer. Cleared lysates were run on 12% SDS-PAGE and blotted with anti-GFP (Santa Cruz B2, Santa Cruz, CA, USA), anti-phospho-Cpi-17 (Santa Cruz-17560, Santa Cruz, CA, USA), anti-Mlc2 (Cell Signaling #3672, Danvers, MA, USA), and anti phospho-Mlc2 (Cell Signaling #3675, Danvers, MA, USA).

Recombinant human TGF-β2 was purchased from Wako Pure Chemical Industries (Osaka, Japan). Y-27632 and SB203580 were purchased from Merck Millipore (Darmstadt, Germany). The anti-COL1A2 (1:2,000 dilution;), anti-Caveolin 1 (1:200 dilution), and anti-myocilin (1:100 dilution) antibodies were obtained from Abcam (Cambridge, UK). The anti-MLC2 (1:800 dilution), anti-phospho-MLC2 (1:700 dilution; Thr18/Ser19), anti-Smad2/3 (1:1,000 dilution), anti-phospho-Smad2 (1:1,000 dilution; Ser465/467), anti-α-tubulin (1:1,000 dilution), and anti-lamin A/C (1:2,000 dilution) antibodies were purchased from Cell Signaling Technology (Danvers, MA). The anti-β-actin mouse monoclonal antibody (1:10,000 dilution;) was obtained from Sigma (St. Louis, MO). The anti-collagen 4 α 5 chain (1:50 dilution) and anti-matrix protein gla (1:400 dilution) antibodies were purchased from Santa Cruz (Dallas, TX). The tissue inhibitor of matrix metalloproteinase 3 antibody (1:1,000 dilution) was obtained from Thermo Pierce (Rockford, IL). The anti-vascular cell adhesion protein 1 antibody (1:100 dilution) was obtained from R&D systems (Minneapolis, MN).

The following antibodies were used: mouse IgG1 (MG100) from Life Technologies (Carlsbad, CA), anti-integrin β1 P5D2 (ab24693) from Abcam (Cambridge, UK), anti-tubulin α (T9026) from Sigma-Aldrich, HRP-conjugated polyclonal goat anti-mouse or anti-rabbit antibodies from DAKO Cytomation (Glostrup, DK), and anti-integrin β1 (#4706), anti-MLC2 (#3672), anti-NMHCIIA (#3403), anti-NMHCIIB (#3404), anti-NMHCIIC (#8189), Alexa Fluor^® 488-conjugated anti-mouse IgG (H+L) antibodies from Cell Signaling Technologies (Danvers, MA).

The following antibodies and working concentrations were used: anti-Actin C11 (1:5000, Sigma Aldrich A2066, for western blot), anti-SREBP1 2A4 (1:500, Santa Cruz Biotechnology sc13551, for western blot), anti-SREBP1 H160 (1:100, Santa Cruz Biotechnology sc8984, for immunofluorescence), anti-SREBP2 (1:500, BD Bioscience 557037, for western blot), anti-SCD1 (1:1000, Abcam ab19862, for western blot), anti-GAPDH (6C5) (1:5000, Santa Cruz Biotechnology sc32233, for western blot), anti-AMPK (1:1000, Cell Signalling 2532S, for western blot), anti-AMPK phospho Thr172 (1:1000, Cell Signalling 2531S, for western blot), anti-ACC1 (1:1000, Cell Signalling 3676S, for western blot), anti-ACC1 phospho Ser79 (1:1000, (Cell Signalling 11818S, for western blot), anti-Farnesyl (1:1000, AB4073 Merck Millipore), anti-Hsp90 (1:2000, Santa Cruz Biotechnology sc13119, for western blot), anti-MLC2 (1:1000, Cell Signalling 3675S, for western blot), anti-MLC2 phospho Ser19 (1:500, Cell Signalling 3671S, for western blot and immunofluorescence), anti-FAK C-20 (1:1000, Santa Cruz Biotechnology sc-558, for western blot) and anti-FAK phospho Y397 (1:1000, Abcam ab81298, for western blot).

Primary antibodies for Western blot include anti–NKA β1 subunit (Upstate, 05-382), anti-occludin (Invitrogen, 71-1500), anti–zo-1 (Invitrogen, 61-7300), anti–zo-2 (Invitrogen, 71-1400), anti-actin (MilliporeSigma, A2066), anti-GAPDH (MilliporeSigma, CB1-001), anti–NKA β2 subunit (Abcam, ab185210), anti-DDK (OriGene, TA50011-100), anti-MRCKα (Santa Cruz Biotechnology Inc., sc-374568), anti-MYPT1 (Cell Signaling Technology, 2634S), anti–phospho-MYPT1 (Thr696, Cell Signaling Technology, 5163S), anti-MLC2 (Cell Signaling Technology, 3672S), and anti–phospho-MLC2 (Ser19, Cell Signaling Technology, 3671S). The primary antibodies for immunofluorescence include anti–occludin Alexa Fluor594 (Invitrogen, 331594), anti–zo-1-Alexa Fluor594 (Invitrogen, 339194), and anti-MRCKα (Thermo Fisher Scientific, PA1-10038). The inhibitor for MRCKα BDP5290 was purchased from Aobious. Myosin inhibitor blebbistatin was purchased from Abcam.

Immunoblots were performed as described previously [9 (link)]. 50–80 μg protein lysate were separated by SDS-PAGE, transferred to nitrocellulose membranes and incubated with the following primary antibodies: anti-α-Tubulin (Sigma, T6199; 1:5000), anti-β-Actin (Sigma, A5441; 1:5000), anti-HA (Roche, 3F10; 1:1000), anti-MLC2 (Cell Signaling Technology, 3672; 1:1000), anti-MYLK (Abcam, EP1458Y; 1:1000), anti-pMLC2 (Cell Signaling Technology, 3674; 1:1000), anti-pPYK2 (Cell Signaling Technology, 3291; 1:1000), anti-TKS5 (Santa Cruz, sc-30122; 1:1000) and anti-ZEB1 (Sigma Prestige, HPA027524; 1:5000).