Protease and phosphatase inhibitors

HRECs were lysed in RIPA extraction buffer supplemented with protease and phosphatase inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA) [83 (link)]. Denatured samples (20 μg) were subjected to SDS-PAGE. Non-specific binding sites were blocked at room temperature for 1 h with 5% (w/v) Blotting-Grade Blocker (Bio-Rad Laboratories, Hercules, CA, USA) in Tris-buffered saline (Boston BioProducts, Boston, MA, USA) containing 0.05% (v/v) Tween-20 (Thermo Fisher, Waltham, MA, USA). Membranes were incubated overnight with the primary antibodies, anti-APEX1 (1:1000) (Novus Biologicals, Centennial, CO, USA; 13B8E5C2), and anti-vinculin (1:1000) (Novus; CP74-100), and then with the peroxidase-conjugated secondary antibody (1:1000) (Bio-Rad, 1706516) for 1 h. Signals were then captured by using a Bio-Rad ChemiDoc imager, and band intensities were analyzed by densitometry using Image Lab software (Bio-Rad).

Cells were kept in RPMI containing 0.5% BSA overnight in an incubator to achieve quiescence, stimulated with SDF-1 (300, 100, or 30 ng/ml), and then lysed for 20 min. on ice with the RIPA lysis buffer system (Santa Cruz Biotechnology, Dallas, TX, USA), containing protease and phosphatase inhibitors (Santa Cruz Biotechnology) as described 5 (link),18 (link). The concentrations of extracted proteins were measured with the BCA protein assay kit, according to the manufacturer’s instructions (Thermo Scientific, Rockford, IL, USA), and equal amounts of protein were separated and analysed for phosphorylation of p44/42 MAPK and AKT (Ser473). Equal loading in the lanes was evaluated by stripping the blots and reprobing with antibodies for p44/42 MAPK and AKT. All phosphospecific antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). The membranes were developed with Amersham ECL Western Blotting Detection reagents and exposed to Amersham Hyperfilm (GE Healthcare).

Cells were lysed in RIPA buffer in presence of protease and phosphatase inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein concentrations were determined by colorimetric assay (Pierce™ BCA Protein Assay Kit, Thermo Scientific). Western blotting was performed using 35–40 µg of protein extracts (60 µg for the detection of LATS1 phosphorylation), using the following primary antibodies: α-tubulin (sc- 32293), Bcl-2 (sc-509) and Bcl-xL (sc-8392) were from Santa Cruz Biotechnology YAP (#12395), phosphorylated YAP (S127, #4911), LATS1 (#3477), phosphorylated LATS1 (#8654), MST2 (#3952), MOB1 (#13730), CTGF (connective tissue growth factor, also known as CCN2, #10,095), vinculin (#13901), pERK1/2 (#9106), ERK (#9102) and H3 (#4499) were from Cell Signaling (Danvers, MA, USA); β-actin (#A1978), alpha-smooth muscle actin, α-SMA (#A5228) was from Sigma-Aldrich; heat shock protein (HSP)72/73 (#HSP01) was from Calbiochem (San Diego, CA, USA). Enhanced Chemiluminescent Substrate method (LiteAblotTURBO, Euroclone) was used to detect immunostained bands, except for the detection of phosphorylated LATS1, by Clarity Max Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA). ChemiDoc System instrument (Bio-Rad Laboratories) was used to acquire images, while ImageJ software was used for densitometric evaluation and normalization with relative controls.

Protein extracts were harvested with protease and phosphatase inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and were analyzed using the following antibodies: C/EBPβ (C-19, Santa Cruz Biotechnology), Smad2/3 (Cell Signaling Technology, Danvers, MA, USA), myogenin, Pax7, and myosin heavy chain (MF-20) primary antibodies from DSHB (Iowa City, IA, USA); and phospho Smad2/3 (Abcam, Cambridge, U.K.). β-Actin (Sigma-Aldrich, St-Louis, MO, USA) was used as a loading control. HRP-conjugated secondary antibodies were from GE Healthcare (Buckinghamshire, U.K.). Chemiluminescence images were captured using the Luminescent Image Analyzer LAS-4000 (Fujifilm Life Science, Tokyo, Japan), and quantifications were done using ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/, 1997–2014).