Anti puma

MTT, dimethyl sulfoxide (DMSO), PI, DAPI and DCFH-DA were purchased from Sigma (St Louis, MO). RIPA buffer and JC-1 staining kit were purchased from Beyotime Biotechnology, China. The following antibodies were used: anti-PUMA, anti-p53, anti-Chk1, anti-Chk2, anti-p-Chk1(Ser345), anti-p-Chk2 (Thr68), anti-ATM, anti-p-ATM (Ser1981), anti-PARP, anti-GAPDH and anti-β-actin (Santa Cruz Technology, Santa Cruz, CA, USA); Anti-NOX1, anti-NOX4, anti-DUOX1 and anti-SOD1, anti-Keap1, anti-Nrf2, anti-HO-1, anti-DNA-PKcs, anti-p-DNA-PKcs (Thr 2609), anti-Caspase-3 and anti-Caspase-9 (Cell Signaling Technology, Danvers, MA, USA); Anti-γ H2AX (Ser139), anti-H2AX and anti-JNK, anti-p-JNK, anti-BCL-XL, anti-MCL-1 and anti-BCL-2 (Abcam). Rhodamine (TRITC) AffiniPure Goat anti-Rabbit IgG was from Santa Cruz Biotechnology; KU-60019, VE-821 and NU7026 were obtained from Selleck (USA). SP600125 were obtained from Calbiochem (La Jolla, CA, USA). Z-VAD-fmk was purchased from R&D Systems (Minneapolis, MN). These inhibitors were dissolved in DMSO and diluted to appropriate concentrations with cell culture media. Mitotracker and MitoSOX red mitochondrial superoxide indicator was purchased from Molecular Probe (USA).

Processed paraffin-embedded synovial sections were stained with anti-GFP (Santa Cruz), anti-PUMA (Santa Cruz), anti-p73 (Santa Cruz), anti-iASPP (Santa Cruz), anti-cadherin-11 (Cell Signaling) or isotype control IgG (Santa Cruz), followed by secondary antibodies and substrate chromogen with expression intensities quantitated by the HistoQuest software (Tissue Gnostics). Apoptotic cells in cryostat synovial sections were detected by the TUNEL assay (Promega). For immunofluorescent analyses, pre-treated paraffin-embedded synovial sections were incubated with anti-p73 (Santa Cruz), anti-iASPP (Santa Cruz), or isotype control IgG (Santa Cruz), followed by FITC- and Texas red-conjugated secondary antibodies (BD Biosciences), respectively, and observed by the fluorescence microscopy. Apoptotic and fluorescence-positive cells were counted by averaging their numbers in 3 randomly selected fields at the ×400 magnification, as previously described [18 (link)]. Purified SFs were subjected to anti-p73 (Santa Cruz) and anti-iASPP (Santa Cruz), followed by Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (Life Technologies), respectively, and observed by the confocal microscopy.

Cell extracts were prepared in RIPA (radioimmunoprecipitation assay) buffer supplemented with Complete protease inhibitor cocktail (Roche). Equal amounts of cell extracts were resolved on acrylamide: bis-acrylamide gels and electroblotted onto PVDF membrane (Immobilon-P, Millipore) and probed with appropriate primary and HRP-conjugated secondary antibodies (Jackson ImmunoResearch). When necessary, membranes were stripped using Restore Western Blot Stripping Buffer (Thermo) and reprobed with anti-PUMA, anti-GAPDH, anti-S100A4, anti-MMP-2/9, anti-bcl-2, anti-bax and cleaved-caspase-3 (Santa Cruz, Shanghai, China). Unless otherwise specified, western blots were representative of n = 3.