Protein a agarose beads

Total protein was extracted from HCT-116 cells and protein concentration was measured using a BCA Protein Assay Kit (KeyGen). Total protein lysate (2 mg) was used for each immunoprecipitation (IP) using specific antibody or the corresponding IgG control. Protein A agarose beads (GE Healthcare, Uppsala, Sweden) were added to the cells and then were incubated overnight at 4 °C. Washed precipitated proteins were analyzed by western blotting. The IP, western blotting and GST pull-down assays were used in this study as described previously in detail.^{40 (link)}

Co-immunoprecipitation samples were obtained by lysing cells in RIPA buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, 1mM EDTA, Protease Inhibitors Cocktail) for 10min on ice. After centrifugation, protein concentration in the supernatants was assayed by Pierce™ BCA Assay, and 0.5-1 mg of pre-cleared protein sample was incubated with 2 μg of antibody as indicated, overnight at 4°C, on constant rotation. Immuno-complexes were precipitated by adding protein A-agarose beads (GE Healthcare) at 4°C for 2hrs. After extensive washes with RIPA buffer, immune-complexes were eluted from the beads by boiling in 1X LDS-buffer +2.5% βmercaptoethanol. Samples were resolved on 4%–12% PAA pre-cast gels and blotted on PVDF membrane (Amersham). For western blotting, cells were rinsed with PBS and lysed in the plates with 1X LDS-buffer +10% βmercaptoethanol. SDS-PAGE and blotting was then performed as described above. For immunodetection, membranes were blocked in 5% milk for 1hr at room temperature and incubated overnight with the indicated antibodies. Antibodies and probing conditions are shown in Table S4.

Cells were lysed with ice-cold radioimmunoprecipitation assay lysis buffer at 4°C for 1 hrs. Samples were subjected to SDS-PAGE, transferred to PVDF membranes (Millipore) and detected using appropriate primary antibodies followed by horseradish peroxidase-conjugated goat anti-mouse or rabbit IgG. The blotting signals were detected using SuperSignal West Dura Extended Duration Substrate (Pierce).
For immunoprecipitation, 1 μg appropriate antibody was preincubated with 30 μl slurry of Protein A-agarose beads (GE Healthcare Life Sciences). Lysates (~1 mg/sample) were incubated with the antibody-bound Protein A-agarose beads at 4°C overnight. After extensive washing with the radioimmunoprecipitation assay lysis buffer, samples were resuspended in the reducing SDS sample loading buffer, boiled for 5 min, and subjected to SDS-PAGE and immunoblotting.

Antibodies against AMPKα1/2, AMPKα2, p‐AMPKα1/2(Thr¹⁸³/Thr¹⁷²), phospho‐ACC (Ser⁷⁹), ACC, and 14‐3‐3 were obtained from Abcam (San Francisco, CA, USA). TBC1D1 and p‐TBC1D1 (Ser²³⁷) were purchased from Merck Millipore (Darmstadt, Germany). Antibodies against HDAC5 and p38 MAPK were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against β‐actin were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Antibodies against phospho‐p38 (Thr¹⁸⁰/Tyr¹⁸²), α‐tubulin, GLUT4, and lamin B were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho‐HDAC5 (Thr⁴⁹⁸) antibodies were from Thermo Fisher Scientific (Rockford, IL, USA). Horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG and goat anti‐mouse secondary antibodies were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Metrnl was obtained from Cusabio (Wuhan, Hubei, China), and 1, 2‐bis (o‐aminophenoxy) ethane‐N, N, N′, N′‐tetraacetic acid (BAPTA)‐AM was purchased from Abcam. Compound C and STO‐609 were obtained from Calbiochem (San Diego, CA, USA). Protein A‐agarose beads were obtained from GE Healthcare (Piscataway, NJ, USA). The fluorescent Ca²⁺ indicator Fluo‐3 AM and Hoechst 33342 were obtained from Invitrogen (Leiden, the Netherlands).

For immunoprecipitation, the cells were washed by cold PBS twice before being lysed in IP lysis buffer supplemented with protease and phosphatase inhibitors. Total protein lysate (2 mg) was used for each immunoprecipitation using specific antibody, Protein A agarose beads (GE Healthcare Uppsala, Sweden) were added to the cells and incubated overnight 4°C. Washed precipitated proteins were analyzed by western blot. The immunoprecipitation (IP), western blot and GST pull-down assays used in this study have been described previously in detail [23 (link)]. The samples were incubated with anti-E-cadherin (BD), anti-PAK5 (R&D), anti-GATA1/-Myc (Santa Cruz), anti-pGATA1 S161 (Bioss), anti-GFP (Genscript), anti-fibronectin (Sigma), anti-Flag (Shang Hai Ruixing), anti-His (Genscript), anti-GAPDH (Kang Chen, as a loading control) antibodies.