Trabecular meshwork cells subjected to various treatments along with their respective controls were homogenized at 4°C in hypotonic 10 mM Tris buffer, pH 7.4, containing 0.2 mM MgCl
2, 5 mM N-ethylmaleimide, 2.0 mM Na
3VO
4, 10 mM NaF, 60 μM phenylmethyl sulfonyl fluoride, 0.4 mM iodoacetamide, and protease and phosphatase inhibitor cocktail (one tablet each/10 ml buffer), using a probe sonicator as we described previously.
31 Protein concentration of cell lysate (800
g supernatants) and conditioned media (CM) samples was estimated using the Bio-Rad protein assay reagent (C. No. 500-0006, Bio-Rad Laboratories). Samples containing equal amounts of proteins (10 μg for the SDS-urea soluble ECM fraction, cell lysates and CM) were mixed with Laemmli sample buffer and separated using 10% to 12% SDS-PAGE and transferred to nitrocellulose membranes as we described previously.
31 Membranes were blocked for 2 hours at room temperature in Tris buffered saline (TBS) containing 0.1% Tween 20 and 5% (wt/vol) nonfat dry milk and subsequently probed with primary antibodies (at 1:1000 dilution) directed against GDF-15, Hic-5 (mouse monoclonal, C.No. 611164; BD Biosciences, San Jose, CA, USA), phospho-paxillin (polyclonal, C.No. 2541, Cell Signaling Technology, Danvers, MA, USA), phospho-SMAD2/3 (polyclonal, C.No. 8828; Cell Signaling Technology), phospho-SMAD1/5 (polyclonal, C.No. 9516; Cell Signaling Technology), phospho-MLC (polyclonal, C.No. 3674; Cell Signaling Technology); MLC (polyclonal, C.No. 3672; Cell Signaling Technology), phospho-MYPT1 (polyclonal, C.No. ABS45; Millipore, Billerica, MA, USA), αSMA (monoclonal, A2547, Sigma-Aldrich Corp.), fibronectin (1:15000 dilution; polyclonal C.No. ab23750; Abcam, Cambridge, MA, USA), and GAPDH (mouse monoclonal antibody at 1:10000 dilution; C. No. 60004-1-g, Proteintech Group, Inc. Rosemont, IL, USA), in conjunction with horseradish peroxidase-conjugated secondary antibodies. Detection of immunoreactivity was based on enhanced chemiluminescence. Densitometric analysis of the immunoblots was performed using ImageJ software (available in the public domain at
http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health (NIH), Bethesda, MD, USA). Data were normalized to the specified loading controls. Urea-glycerol gels were used for phospho-MLC immunoblots, as we described previously.
33