Anti iaspp

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.

Lysates of SFs or extracts from synovial tissues were subjected to immunoblot with anti-p73 (Santa Cruz), anti-GFP (Santa Cruz), or anti-cadherin-11 (Cell Signaling), and the blots were re-probed with anti-β-actin (Sigma-Aldrich) as a quantitative control, as previously described [8 (link), 20 (link)]. AdLacZ or Ad37AA-transduced SF lysates were immunoprecitated with anti-iASPP (Santa Cruz), followed by immunoblotted with anti-p73 (Santa Cruz) or anti-GFP (Santa Cruz), and small quantities (10%) were snapped for immunoblot analysis as an input control.

Total cell lysates were extracted using the M-Per Mammalian Protein Extraction Reagent (Pierce Biotechnology, MA, USA), followed by immunoblotting with anti-iASPP (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phosphorylated p53 (1:1000; Abcam, Cambridge, UK), anti-Caspase-3 (Cell Signaling Technology, Danvers, MA, USA), anti-E-cadherin (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-Vimentin (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-FBXL5 (1:1000; Abcam, Cambridge, MA, USA), anti-BTG3 (1:1000, Abcam, Abcam, Cambridge, UK) or anti-GAPDH (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were incubated with appropriate secondary antibodies and immunoreactive bands were visualized with ECL reagent (Perkin Elmer, Waltham, MA, USA). GAPDH was used as the endogenous control.