Protein a g sepharose beads

After the treatment with metformin (2 mM), glucose (15 mM) and/or siAMPK (50 nM)/siControl (50 nM) or Compound C (1 μM) at 24 h or 48 h, the cells were lysed in ice-cold NP-40 buffer containing protease inhibitor (1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride). The whole-cell lysates were incubated with protein A/G Sepharose beads (Roche) for 2 h at 4 °C, and the beads were discarded to eliminate non-specific binding. Then, the supernatants were incubated overnight with a Bcl-xl antibody (Cell Signaling Technology, Danvers, USA) at 4 °C, followed by incubation with protein A/G Sepharose beads for another 2 h at 4 °C. Then, western blotting was performed with the indicated antibodies according to standard protocols as previously described^{20 (link)}. The primary antibodies, including p-AMPK (T172) (#50081), phospho-Acetyl-CoA carboxylase (p-ACC) (#11818), phospho-Tuberin/TSC2 (p-TSC2) (Ser1387) (#23402), phospho-Raptor (p-Raptor) (Ser792) (#2083), p-mTOR (S2448) (#5536), phospho-p70 S6 kinase (p-S6K1) (Thr389) (#97596), p-ULK1 (Ser757) (#14202), p-4E-BP1 (Thr70) (#9455), cleaved Caspase-3 (Cl-Caspase3) (Asp175) (#9664), p-Akt (Ser473) (#4060), p-Akt (Thr308) (#13038), RagB (#8150), LC3B (#3868), β-Actin (#3700) were all purchased from Cell Signaling Technology, Danvers, USA.

Interactions between IER3 and TRAIL were assessed via co-IP assays. Cells were first lysed using RIPA buffer after being washed with PBS. Lysates were then mixed for 12 h with appropriate primary antibodies linked to protein A/G sepharose beads (Roche, shanghai, China) at 4 °C following protein G sepharose bead pre-clearing. SDS-PAGE was then used to separate precipitated immunocomplexes, and separated proteins were transferred to PVDF membranes (Millipore, Bedford, MA, USA). Blots were then blocked for 2 h using 5% non-fat milk in TBST, followed by an overnight incubation with antibodies specific for TRAIL (1:1,000; Santa-Cruz Biotechnology, Santa Cruz, CA, USA), IER3 (1:1,000; Sigma, St. Louis, MO), Myc tag (1:1,000; Sigma, St. Louis, MO), and Flag tag (1:1,000; Cell Signaling, Danvers, MA) at 4 °C. Blots were next washed thrice, followed by incubation for 2 h with appropriate HRP-linked secondary anti-rabbit and anti-mouse antibodies (1:1,000; Pierce, USA). A Bio-Rad ChemiDoc Imaging System (Bio-Rad, California, USA) was then employed for protein band detection.

Cells were lysed in NP-40 lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 10 mM phenylmethylsulfonyl fluoride (PMSF), 20 mM NaF, 1 mM Na₃VO₄ and a protease inhibitor ‘cocktail’ (Sigma). Lysates were immunoprecipitated with the appropriate antibodies for 4 h at 4°C. Protein A/G Sepharose beads (Roche) were then added, and the samples were incubated overnight. Alternatively, lysates were immunoprecipitated with anti-Flag M2 Magnetic beads (Sigma) for 4 hours at 4°C. After three washes, samples were resolved on SDS-PAGE gels (10% or 12%) and blotted. For immunoblot analysis, cells were lysed in SDS sample buffer by the addition of 1/4 volume of 5×SDS sample buffer directly into the cell suspensions. Samples were then boiled for 5 min and separated using 10% or 12% SDS-PAGE.

Cells were lysed in ice-cold NP-40 buffer containing protease inhibitor (as above). The whole-cell lysates were incubated with protein A/G Sepharose beads (Roche) for 2 h at 4 °C and the beads were discarded to eliminate the non-specific binding. Then, the supernatants were incubated overnight with DOT1L antibody at 4 °C, followed by incubating with protein A/G Sepharose beads for another 2 h at 4 °C. Afterward, western blotting with indicated antibodies was performed. Standard western blotting protocols were adopted. Relative protein levels were quantified by measuring the band intensity of western blots by using Image J software. All the uncropped, full-size scans of western blots are presented in Supplementary Fig. 14.

Immunoprecipitated the cell lysates with anti‐SP1 antibody followed by protein A+G‐Sepharose beads (Roche) at 4°C overnight. The beads were washed with lysis buffer. Resuspend the pellet in the same volume of SDS sample buffer and boiled to remove the Sepharose beads. The whole cell lysates and immunoprecipitated proteins were then analysed by WB.

After transfection and stimulation, HEK293T cells were harvested and lysed with a lysis buffer for 30 min at 4°C. Cell lysates were centrifuged at 12,000 rpm for 10 min, and the supernatant (1 μg total protein) was incubated with the indicated antibodies (1 μg) and protein A/G-Sepharose beads (Roche) for 2 h at 4°C. The beads were then washed 5 times with the lysis buffer. The beads with a loading buffer were boiled for 10 min at 100°C and subjected to SDS-PAGE.