Anti acetylated histone 3

Cell lysates of SFs or synovium homogenates were subjected to immunoblot with anti-Smad7 (R&D systems), anti-Flag (Sigma-Aldrich), anti-Snail (Cell Signaling), anti-Cadherin-11 (Cell Signaling), anti-acetylated-histone 3 (Cell Signaling) or anti-β-actin (Sigma-Aldrich), followed by secondary antibodies, developed with ECL Plus system (Amersham), analyzed by Biospectrum imaging system (UVP), and quantitated for the signaling intensities by ImageJ software (National Institutes of Health), as previously described9 (link)18 (link). Expression of acetylated Smad7 in TSA (Sigma-Aldrich)-treated SFs lysates was immunoprecipitated by anti-Smad7 (Santa Cruz), followed by anti-acetylated-lysine (Cell Signaling) immunoblot with small quantities (~10%) snapped for immunoblot analysis as an input control.

Normal GC B cells, DLBCL cell lines, or primary DLBCL specimens were lysed using RIPA lysis buffer containing complete protease inhibitor cocktail to prepare whole-cell lysate. Whole-cell lysates were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane (Bio-Rad), and probed with the indicated primary antibodies: anti-SIRT3, anti-ATF4, anti-GRP75, anti–acetylated histone 3, anti-LC3, and anti-p62, purchased from Cell Signaling Technology, and anti-ACTB, anti–histone 3, and anti-GDH, purchased from Sigma-Aldrich, Millipore, and Proteintech Group, respectively. Membranes were then incubated with a corresponding peroxidase-conjugated secondary antibody. Protein signals were detected using enhanced chemiluminescence. Densitometry values were obtained by using ImageJ 1.44o.

Cells were harvested, washed with PBS, and lysed with lysis buffer supplemented with protease inhibitors (Roche, Sainte-Agathe-Nord, QC, Canada). Protein concentrations were determined using a Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA), and samples were then normalized. The samples were loaded into an 8–10% polyacrylamide gel and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transference to a polyvinylidene difluoride membrane. Proteins were identified by incubation with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence solution (Pierce, Thermo Scientific, Waltham, MA, USA). Antibodies used in this study include: anti-phosphorylated S6 Ribosomal Protein (1:1000, Cat: 2211s, Cell Signaling, Cambridge, MA, USA), anti-S6 Ribosomal Protein (8E2) monoclonal antibody (1:1000, Cat: 2217s, Cell Signaling), anti-4E-EBP1 (1:1000, Cat: 9452s, Cell Signaling), anti-phosphorylated 4E-BP1 (1:1000, Cat: 2855s, Cell Signaling), anti-acetylated Histone 3 (1:1000, Cat: 4243s, Cell Signaling), anti-Histone 3 1:1000, Cat: 9715s, Cell Signaling), anti-ESR1α (1:1000, Cat: MCA1799T, Bio Rad, CA, USA), and anti-α-tubulin monoclonal antibody (1:500, Cat: T9026, Sigma-Aldrich, St. Louis, MO, USA).