A5228

HFD and CD ASCs cells were grown to confluency on 10-cm plates in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic. Media were removed, and cells were washed with PBS twice. Proteins were extracted in RIPA buffer including protease and phosphatase inhibitors. Electrophoresis was performed with 4%-20% gel (Bio-Rad, 456-8093) with Tris/Glycine/SDS running buffer (Bio-Rad, 161-0772). Proteins were transferred to Amersham Hybond-ECL membrane (GE Healthcare, RPN303D). Membranes were blocked for 1 hour with 5% dry milk powder and 1% BSA (Sigma, A4503) in TBST. Membranes were probed for antibodies against SMA (Sigma-Aldrich, A5228, 1:5000), collagen I (abcam, ab34710, 1:5000), IGF-1 (R&D Systems, AF791, 1:250), or GAPDH (Invitrogen, MA5-15738, 1:5000). Secondary antibodies conjugated to horseradish peroxidase were goat anti-rabbit IgG (Invitrogen, 31460, 1:10,000), goat anti-mouse IgG (Invitrogen, 31430, 1:10,000), or rabbit anti-goat IgG (Invitrogen, 31402, 1:5000). Detection substrate used was SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, 34080) or Clarity Western ECL Substrate (BioRad, 1705060). Amersham Hyperfilm ECL (GE Healthcare, 28-9068-38) film was used for development on the All-Pro Imaging Corp 100 Plus Automatic X-Ray Film Processor (All-Pro).

Proteins were extracted from TGF-β1-stimulated oral myofibroblasts with RIPA buffer (20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 50 mM b-glycerophosphate, 150 mM NaCl, 1 mM Na3VO4, 1% NP-40, 25 mM NaF) containing protease and phosphatase inhibitors cocktail (Sigma-Aldrich). The solubilised proteins were resolved using SDS-PAGE and transferred onto polyvinylidene fluoride membranes (ATTO, Tokyo, Japan). Protein identification was performed by western blotting using primary antibodies raised against αSMA (1/1000; Sigma, A5228) and GAPDH (1/5000; Abcam ab185059), diluted in TBS containing 5% dried milk and 3% BSA, followed by secondary antibodies conjugated with horseradish peroxidase. Immunoreactive bands were detected using enhanced chemiluminescence reagents (GE Healthcare; Pittsburgh, PA, USA).