Protein a g plus agarose slurry

HEK293 cells were co-transfected with plasmids encoding Xpress-AtNAA15 and AtNAA10-V5 using FuGENE6 (Roche), harvested 48 h post transfection and lysed in 1 ml IPH buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% NP-40, 5 mM EDTA, 1 mM Na₃VO₄, 2 mM pefabloc and 1 × complete protease inhibitor cocktail (Roche)). Approximately 1 × 10⁷ cells were used for each sample. Protein A/G Plus-Agarose slurry (50 μl, Santa Cruz Biotechnology, Santa Cruz, USA) was added and incubated for 1 h at 4 °C. After centrifugation at 500g for 5 min, the supernatants were incubated with 3.0 μg anti-V5 mouse monoclonal IgG_2a ab (Invitrogen), anti-Xpress mouse monoclonal IgG₁ ab (Invitrogen) or unspecific IgG_2a ab (DAKO) and incubated for 4 h at 4 °C. Later, 70 μl of Protein A/G Plus-Agarose was added and incubated for 16 h at 4 °C with the samples. The immunoprecipitate samples were collected by centrifugation at 500g for 5 min, washed four times with 1 × PBS.

Immunoprecipitation (IP) was performed as previously described [9 (link)]. In brief, 500 μg of total cellular proteins from different treatment groups was incubated with 1 μg primary antibodies against ANT and phospho-GSK-3β for 1 h at 4°C. The mixture was incubated with 20 μL of protein A/G PLUS-agarose slurry (Santa Cruz, CA, USA) at 4°C overnight. The samples were subsequently centrifuged at 2500 rpm for 5 min at 4°C. The precipitations were recovered and washed with PBS buffer for 3 times. Finally, the pellets were dissolved in 60 μL of 1x electrophoresis sample buffer and boiled for 5 min. Thirty microliters of each sample was analyzed by Western blotting.