Protein g sepharose

Briefly, P19 or HEK293T cells were cultured in DMEM containing 15% heat-inactivated FBS (Hyclone, Waltham, MA, USA), after transfection cells were lysed in modified RIPA buffer for 30 min at 4 °C with rotation. Two-hundred milligram of cell lysate was pre-cleared with Protein G-Sepharose (Roche, Indianapolis, IN, USA) for 1 h, and then was incubated with anti-FLAG conjugated agarose beads (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C with rotation. Agarose beads were washed with TBS buffer and denatured samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis electrophoresis. Western blot was performed as described above.

To test for interactions between BAG5 domains and PINK1 domains in vitro, binding assays were performed. An aliquot of protein from the soluble fraction of E. coli crude extract were incubated with 30 µl of glutathione agarose beads (Pharmacia) for 30 min on ice. After washing three times with 1×PBS, beads bound with GST-BAG5 or GST-PINK1 were incubated with 50 µg of protein from the supernatants of E. coli crude extract containing recombinant His-PINK1 or His-BAG5 expressed by pET-21a-PINK1 or pET-21a-BAG5 in 0.25 ml HNTG-buffer [20 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 0.1% Triton X-100 and 10% glycerol] for 2 h at 4°C. Samples were washed twice with 1×PBS, followed by addition of His-PINK1 or His-BAG5 for 2 h on ice. Bound proteins were eluted from the beads by boiling in SDS sample buffer and detected by immunoblot analysis. Crude cell lysates were sonicated in TSPI buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mg/ml aprotinin, 10 mg/ml of leupeptin, 0.5 mM Pefabloc SC, 10 mg/ml of pepstatin, 1% NP-40]. Cellular debris was removed by centrifugation at 12 000 g for 20 min at 4°C. The supernatants were incubated with the anti-bodies in 0.01% BSA for 4 h at 4°C. After incubation, protein G Sepharose (Roche) was used for precipitation. The beads were washed with TSPI buffer seven times, and proteins were eluted with SDS sample buffer for immunoblot analysis.

Conditioned media from A549 and H1299 cells were collected and incubated with anti-Hsp90α antibody (Abcam, Cambridge, MA, USA, ab2928, 1:2,000 dilution), anti-GRP78 antibody (Santa Cruz, Dallas, TX, USA, SC-1050, 1:1,000 dilution), and protein G-Sepharose (Roche, Basel, Switzerland) overnight at 4°C with constant rotation. Before analyzing by immunoblot, we washed immuno-complexes three times with IP lysis buffer. Antibodies against PKM2 (#4053, 1:1,000 dilution) and β-actin (#3700, 1:1,000 dilution) were obtained from Cell Signaling Technologies (Danvers, MA).