Pierce coomassie plus bradford assay

Parotid glands were harvested and snap-frozen from untreated and irradiated mice at day 8 or 30 post-IR. Tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer, with protease inhibitor cocktail (30 μL/mL; Sigma, no. P8340), sodium orthovanadate (5 mM) and phenylmethylsulfonyl fluoride (PMSF, 1 mM). Tissue homogenates were incubated on ice for 30 min, sonicated for 1–2 min and centrifuged at 12,000 RPM for 10 min at 4°C to remove cell debris. Cells or aggregates were collected by scraping, centrifuged, resuspended in tissue protein extraction reagent (T-PER, Thermo no. 78510) with protease inhibitor cocktail (30 μL/mL), sodium orthovanadate (5 mM), and PMSF (1 mM), incubated on ice for 10–15 min and centrifuged at 12,000 rpm for 10 min at 4°C to remove cell debris. Protein content was measured with the Pierce Coomassie Plus Bradford assay (Thermo, no. 23236, Waltham, MA, United States) or the Bicinchoninic acid (BCA) assay (Thermo, no. 23225).

hBMSCs were seeded until reaching 60-80% confluence before incubation in serum reduced medium (0.2% FBS), low glucose MEM medium for 6 hours prior to treatment with 1μM of Apigenin, Rutaecarpine or DMSO-vehicle control in osteogenic induction media. Protein samples were harvested at 0, 30, 60, 120 & 240 minutes after treatment. Briefly, cells were washed in PBS and were lysed in RIPA buffer (Invitrogen) supplemented with protease inhibitors (Roche). After 30 min incubation at 4°C, samples were centrifuged for 10 min at 12,000 rpm, 4°C. Protein concentration was determined using Pierce Coomassie Plus Bradford assay (Thermo Fisher Scientific), and equal amounts of proteins were loaded on a 10% polyacrylamide gel (Invitrogen). Blotted nitrocellulose membranes were incubated overnight with antibodies against p-FAK, FAK, p-ERK, ERK2, p-SMAD2, SMAD2 & Actin (Cell Signaling) at 4°C. Membranes were incubated with HRP conjugated anti-mouse or anti-rabbit secondary antibody (Santa Cruz Biotechnology) for 45 min at room temperature, and protein bands were visualized with Amersham ECL chemiluminescence detection system (GE Healthcare Bio-Sciences Corp).

Histone H3 and H4 acetylation were quantified using the EpiQuik™ Global Histone H3 Acetylation Assay Kit (Epigentek, Farmingdale, NY, USA) and the EpiQuik™ Global Histone H4 Acetylation Assay Kit (Epigentek, Farmingdale, NY, USA) according to the manufacturer’s instructions. First, histones were isolated from freshly isolated PBMCs using the reagents of the respective kit as well as trichloroacetic acid (TCA) and acetone. Histones were then quantified using the Pierce™ Coomassie Plus Bradford Assay (Thermo Scientific™, Waltham, MA, USA). Histone extracts were stored at −80 °C until assayed. Subsequently, 1 ug of protein was immobilized on a 96-well plate and acetylated H3/H4 was detected with capture and detection antibodies. The plate was read at 450 nm using the FlexA-200 microplate reader (Allsheng, Hangzhou, China), and OD was displayed directly.