Irdye 800 goat anti rabbit antibody

After treatment with various cytokines and inhibitors, MSCs were washed with cold PBS and lysed with RIPA lysis buffer (Boster) plus protease inhibitor cocktail (Thermo) and phosphatase inhibitor tablet (Roche). Equal amounts of protein lysates were resolved on 10% SDS-PAGE and transferred onto nitrocellulose (NC) membrane (Pall), followed by blocking with 5% skimmed milk in TBST (150 mM NaCl, 0.1% Tween 20, 25 mM Tris-HCl, pH 7.6) for 2 h at room temperature. The membrane was incubated with the primary antibodies overnight and washed with TBST. After incubating with secondary IRDye 680 goat anti-mouse or IRDye 800 goat anti-rabbit antibodies (LI-COR Biosciences), the membrane was detected using the Odyssey Infrared Imaging System (LI-COR Biosciences). The following primary antibodies were used: rabbit COX-2 antibody (Cayman Chemical, 160107), rabbit mPGES-1 antibody (Cayman Chemical, 160140), rabbit S6K1 antibody (Cell Signaling Technology, 9202), rabbit phospho-S6K1 (Thr389) antibody (Cell Signaling Technology, 9234), rabbit 4E-BP1 antibody (Cell Signaling Technology, 9644), rabbit phospho-4E-BP1 (Thr37/46) antibody (Cell Signaling Technology, 2855), mouse Akt antibody (Cell Signaling Technology, 2920), rabbit phospho-Akt (Ser473) antibody (Cell Signaling Technology, 4060), rabbit phospho-GSK-3β (Ser9) antibody (Abcam, ab75814), and mouse β-actin antibody (Sigma, A5441).

Cells were seeded at a density of 1 × 10⁶ cells onto in six-well plates (Sigma-Aldrich) and incubated with 100 µM PT, OT and AM as above. After the incubation, the cells were washed with ice-cold PBS and processed as described by us^{13 (link)}. Immunostaining of phosphorylated p53 at Ser9, Ser20, and Ser392 on nitrocellulose membranes (1:1000, GE Healthcare) was performed using the phospho-p53 antibody sampler kit (Cell Signaling Technology, 9919) followed by secondary detection using IRDye-800 goat anti-rabbit antibody (1:5000, Licor Biosciences, 925–32211) and visualized using the Licor Odyssey Imaging system (Licor Biosciences).

Western blotting analysis was performed as previously reported. Briefly, cells were lysed in immunoprecipitation lysis buffer (Beyotime Biotechnology, Shanghai, China) with 1 mM PMSF on ice for 30 min. Protein concentrations were measured by the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). Equal amounts of protein were separated by SDS-PAGE and transferred onto 0.22 μm nitrocellulose membranes (Millipore, Cork, Ireland). The membranes were incubated with anti-SPP1 (Abcam, ab214050), anti-GAPDH (ABclonal Biotechnology, Hubei, China, A19056), anti-CD44 (Abcam, ab189524), anti-SMAD2/3 (Abcam, ab202445), anti-pSMAD2/3 (Abcam, ab254407), anti-STAT3 (Abcam, ab68153), and anti-pSTAT3 (Abcam, ab76315) overnight at 4 °C, and then incubated with IRDye 800 goat anti-rabbit antibody (LI-COR Biosciences, Lincoln, USA) for 1 h at room temperature. After washing off the unbound antibodies, the labeled bands were scanned by Odyssey® CLx Infrared Imaging System (LI-COR Biosciences, MA, USA).