Protein g sepharose 4b beads

Immunoprecipitation (IP) was performed as described previously [27] (link), [34] (link), [35] (link) with minor changes. Frozen hearts were pulverized under liquid nitrogen and then the powder was lysed in RIPA buffer containing 50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS and protease inhibitors on ice for 30 min. The sample was pre-cleared with 30 µl protein G Sepharose-4B beads (Invitrogen) for 1 h at 4°C with constant end-over-end shaking and then centrifuged at 3000 g for 5 min. Supernatant was collected, adjusted to 2 mg/ml protein concentration with RIPA buffer containing protease inhibitors, and subjected to IP with an anti-Shc antibody (rabbit polyclonal IgG, Millipore) at 4°C with constant end-over-end shaking overnight. The next day 30 µl of protein G Sepharose-4B beads was added and the mixture was incubated for another 2 h under the same conditions as above. The beads were collected and washed (five times) with RIPA buffer. After washing and aspirating the washing buffer completely, 50 µl Laemmli sample buffer containing 50 mM Tris-Cl, 10% glycerol, 500 mM β-mercaptoethanol, 2% SDS, 0.01% w/v bromophenol blue and protease inhibitors at pH 7.4, was added to the beads and boiled at 95°C for 5 min. The immunoprecipated proteins were separated using SDS-PAGE and then subjected to Western blot analyses.

For the AIBP/AIBP^(ΔMLS) and TIM23 coIP, 1–2 × 10⁶ 293 T cells were plated in 6-cm dishes and transfected with 3 μL of Lipofectamine 2000 (Cat# 11668019, Invitrogen) according to the manufacturer’s protocol. The AIBP/TIM23 coIP was performed using cells transfected with 0.25 μg of AIBP-FLAG/AIBP^(ΔMLS)-Flag, 1 μg of PINK1-HA [16 (link)], and the corresponding empty vector controls. Twenty-four hours after transfection, the cells were washed twice with PBS, lysed in 500 μL of coIP buffer (150 mM NaCl, 25 mM HEPES, 0.2% NP40, and 10% glycerol) containing 1 mM DTT and protease inhibitors (1 mM PMSF, 1 μg/mL aprotinin, 10 μg/mL pepstatin and 1 μg/mL leupeptin), incubated for 20 min on ice and clarified by centrifugation at 15,000 rpm for 15 min at 4 °C. A sample was aliquoted as the input, and the remaining lysate was incubated for 6 h at 4 °C with 0.5 μg of anti-FLAG antibody (Cat# F7425, Sigma–Aldrich) or anti-HA antibody (Cat# 2367, Cell Signaling) and overnight with 10 μL of washed Protein G Sepharose 4B beads (Cat# 101242, Invitrogen). The beads were washed with coIP buffer and resuspended in 2X Laemmli buffer. Samples were fractionated on SDS–PAGE gels and analyzed using Western blotting.