Protein a g conjugated beads

For co-immunoprecipitation of SIRT1, SIRT3, or SOD2 complexes, SIRT1, SIRT3, or SOD2 were immunocaptured from mitochondrial extracts, respectively. Lysate of mitochondrial fraction (1 ml) containing 1.5 mg total protein was incubated with 1 mg SIRT1, SIRT3, or SOD2 antibody, respectively, and 20 ml protein A/G-conjugated beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight. After four washes in TNES buffer, samples were centrifuged at 3000 × g for 2 min and resuspended in 20 ml SDS-sample buffer (0.5M Tris–HCl, pH6.8, 20% glycerol, 2% SDS, 5% 2-mercaptoethanol, 4% bromophenol blue). For western blot analysis, 10 ml samples were used. The immunocomplexes were analyzed by western blotting and probed with antibody against anti-HIF1α, or acetylated-lysine antibody (Cell Signaling Technology, Danvers, MA, USA), respectively, and incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology).

Cells were scraped from culture dishes and centrifuged to pellet cells. Cell pellets were lysed on ice for 1 h in immunoprecipitation (IP) buffer (250 mM NaCl, 50 mM Tris, 0.5 mM EDTA, 0.5% NP-40) with protease/phosphatase inhibitor added. For the anti-VP16 immunoprecipitation, 10 mM N-Ethylmaleimide was added to the IP Buffer immediately before use. Lysates were centrifuged to remove cell debris and the soluble portion was precleared with Isotype control antibody (Santa Cruz) and protein A/G conjugated beads (Santa Cruz) for 1 h with agitation at 4 °C. Beads were pelleted by centrifugation and lysates were moved to new tubes. The lysates were then incubated with isotype or specific antibody (5 µg per 1.7 mL tube with 1 mL lysate) on ice for 1 h. 20 µL of protein A/G beads were added and samples were agitated at 4 °C overnight. The beads containing the immunoprecipitated proteins were pelleted by centrifugation and the unbound portion decanted. The beads were washed four times in IP buffer prior to processing for immunoblot analysis.

To examine the TAK1 activity, an in vitro kinase assay was modified from Yang et al. [28 (link)]. The wild type TAK1 and mutant TAK1 from pCMV-HA-TAK and pCDH-TAK1-mut plasmids were subcloned into pcDNA3.1(+)/3xFlag vector to generate pcDNA/Flag-TAK1 and pcDNA/Flag-mutTAK1. Both plasmids were transfected into A2780cp cells, respectively an cell lysates were harvested after 48 hours and treated with Human IL-1α (Peprotech, Rocky Hill, NJ). Cell lysates were prepared by Cell Lysis Buffer (Cell Signaling) and immunoprecipitated with anti-Flag (Sigma) followed by overnight incubation with protein A/G-conjugated beads (Santa Cruz) at 4⁰C. After 3 times of washing using Cell Lysis Buffer, an in vitro kinase reaction was performed at 30°C for 90 min in 20 μl Kinase Buffer (Cell Signaling) containing ATP (200μM) and 1 μg of unactive MKK6/SKK3 protein (Millipore, Billerica, MA). The activities of wild type TAK1 and mutant TAK1 were determined by the levels of phospho-MKK6 using Western blotting.