Mouse anti fibronectin

After treatments, cellular proteins were extracted with ice-cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCL, pH 7.5, 150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 25 mM NaF, 0.1 mM sodium orthovanadate, 10 mM NaP₄O₇, 1 nM phenylmethyl sulfonyl fluoride) containing protease inhibitors (Complete Protease Inhibitor; Roche, Manheim, Germany) on ice. Total protein concentrations were quantified by a bicinchoninic acid assay (Thermo Fischer Scientific, Waltham, MA, USA). A total of 10 μg of proteins from each sample were separated by SDS polyacrylamide gel electrophoresis on a 4% to 12% gel in MOPS running buffer (Life Technologies, Carlsbad, CA, USA), transferred onto a polyvinylidene fluoride (PVDF) membrane and probed with the following primary antibodies: rabbit anti-MMP14, mouse anti-fibronectin, rabbit anti-collagen IV, and mouse anti-GAPDH (Abcam, Boston, MA, USA). HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Invitrogen, Carlsbad, CA, USA) were used. Bound antibody complexes were detected using FluorChem E (ProteinSimple, San Jose, CA, USA). Protein expression was analyzed by densitometry using ImageJ (National Institutes of Health [NIH], Bethesda, MD, USA), and normalized to the housekeeping protein, GAPDH. All experiments were performed in triplicate for each of four donor cells.