Pierce ip buffer

For immunoprecipitation experiment, cells were lysed by ice-cold TX-100 lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM DTT, 1% Triton X-100 [MP bio, 9002-93-1]) supplemented with protease and phosphatase inhibitors. Gently sonication was used to completely lyse cells and shear DNA. For co-immunoprecipitation, cells were prepared in pre-cold Pierce™ IP buffer (Thermo Fisher Scientific, 87787) supplemented with protease inhibitors. The cell lysate was gently rotated at 4°C for 4 h (HulaMixer, Invitrogen) and then centrifuged at 16,000 g for 15 min at 4°C. The indicated antibodies were incubated with the supernatants of cell lysates and gently rotated overnight at 4°C, subsequently mixed with pre-washed protein A/G-plus agarose beads (Santa Cruz Biotechnology, sc-2003) at 4°C for 4 h. Immunoprecipitates were isolation by centrifuging samples at 1,000 g for 3 min and washed 5 times with the lysis buffer before being separated by SDS-PAGE and immunoblotted with indicated antibodies.
For transient transfection experiments, HEK293T and HeLa cells were seeded in culture dishes and incubated for approximately 24 h. Cells were then transfected with indicated pcDNA3.1-based expression vectors by Lipofectamine 2000 transfection reagent for another 24 h.

For immunoprecipitation experiment, cells were lysed by ice-cold TX-100 lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM DTT, 1% Triton X-100 [MP bio, 9002-93-1]) supplemented with protease and phosphatase inhibitors. Gently sonication was used to completely lyse cells and shear DNA. For co-immunoprecipitation, cells were prepared in pre-cold Pierce™ IP buffer (Thermo Fisher Scientific, 87787) supplemented with protease inhibitors. The cell lysate was gently rotated at 4°C for 4 h (HulaMixer, Invitrogen) and then centrifuged at 16,000 g for 15 min at 4°C. The indicated antibodies were incubated with the supernatants of cell lysates and gently rotated overnight at 4°C, subsequently mixed with pre-washed protein A/G-plus agarose beads (Santa Cruz Biotechnology, sc-2003) at 4°C for 4 h. Immunoprecipitates were isolation by centrifuging samples at 1,000 g for 3 min and washed 5 times with the lysis buffer before being separated by SDS-PAGE and immunoblotted with indicated antibodies.
For transient transfection experiments, HEK293T and HeLa cells were seeded in culture dishes and incubated for approximately 24 h. Cells were then transfected with indicated pcDNA3.1-based expression vectors by Lipofectamine 2000 transfection reagent for another 24 h.

Differentiated adipocytes were lysed in Pierce IP Buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitors (Roche Life Science). Cell debris was pelleted at 20,000 × g for 15 min at 4°C. After removal of the lipid supernatant, 10 mg of protein from the infranatant was incubated overnight on an end-over-end rotator at 4°C with antibodies directed against the FLAG-tag (conjugated to magnetic beads, Sigma Aldrich, 20 μL of packed gel volume), ATP synthase, beta subunit (10 μg, Abcam, ab128743), or the C-terminal Myc tag of HADHB (10 μg, Abcam, ab9106) (Table 1). Pre-immune immunoglobulin immunoprecipitations (IPs) were performed as controls with equal quantities of protein, using magnetic bead-conjugated mouse immunoglobulin G (IgG) (Cell Signaling Technology) or rabbit IgG (Santa Cruz). After overnight incubation, ATP synthase and HADHB IP reactions and rabbit IgG control reactions were incubated with protein A-conjugated magnetic beads (25 μL, Thermo Scientific) for 1 h at room temperature on an end-over-end rotator. Bead-protein complexes were pelleted on a magnetic stand and washed with Tris-buffered saline ten times. Protein was eluted from the beads using 100 μL of 0.1 M glycine (pH 3.0). Samples were equilibrated to neutral pH by addition of 1 M triethylammonium bicarbonate (TEAB) buffer (pH 8.0) to a final concentration of 100 mM TEAB.