C myc peptide

For the in vitro immunoprecipitation assays using purified proteins, we first transfected SP/HA tagged K3 or R3 human HIF-2α[8 (link)], c-myc tagged WT or HAT mouse CBP, or V5 tagged WT or MUT mouse Acss2 expression constructs into HEK293T cells; waited 48 hr; prepared whole cell extracts; incubated the ectopic HIF-2α, CBP, or Acss2 transfected cell extracts with HA (Cat. No. H9658, Sigma), c-myc (Cat. No. sc-789, Santa Cruz Biotechnology), or V5 (Cat. No. 46–0705, Invitrogen) antibodies, respectively; and then purified the ectopic proteins with protein A/G agarose (Cat. No. sc-2003, Santa Cruz Biotechnology). The ectopic proteins were eluted with 10 μg/mL working solutions of soluble competitor HA peptide (Cat. No. I2145, Sigma), c-myc peptide (Cat. No. M2435, Sigma), or V5 peptide (Cat. No. V7754, Sigma). For the in vitro immunoprecipitation assays, each reaction mixture contained 60 mM potassium phosphate (pH 7.5), 0.1 mM CoA, 4 mM MgCl₂, 1 mM DTT, without or with 3 mM ATP plus 0.24 mM sodium acetate where indicated, in addition to purified CBP, Acss2 and HIF-2α ectopic proteins as specified. Where indicated, acetyl CoA was added to the final concentration instead of ATP and sodium acetate. After addition, we incubated the mixture for 30 min at 30°C, performed pulldown, and subjected the bound proteins to immunoblot analyses as indicated.

Nuclear protein extracts were prepared, as described [69 (link)], from inducible MCF-7 Tet-On cells expressing Myc-Flag-ERβ treated or not with doxycycline (2 μg/ml) for 24 h. To immunoprecipitate AGO2, 1 mg of nuclear proteins was incubated overnight at 4 °C with 2 μg of mouse monoclonal anti-AGO2/eIF2C2 (ab57113, Abcam) and then at 4 °C for 1 h with 35 μl of equilibrated slurry Protein A/G Plus-Agarose (sc-2003, Santa Cruz Biotechnology). To immunoprecipitate myc-flag-tagged ERβ, the same amount of nuclear proteins was incubated for 2 h at 4 °C with 35 μl of equilibrated slurry EZview Red Anti-c-Myc Affinity Gel (E6654, Sigma Aldrich). After binding, the beads were sequentially washed with IPP150 buffer (7.14 mM HEPES pH 7.5, 8.92% glycerol, 150 mM NaCl, 0.54 mM MgCl₂, 0.07 mM EDTA pH 8, 1× protease inhibitors) and wash buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1× protease inhibitors). To elute ERβ-immunoprecipitated samples from the beads, an elution at 4 °C for 30 min was performed using c-Myc Peptide (M 2435, Sigma Aldrich).

To investigate detergent solubilisation conditions for the immunoprecipitation of TbGPI3-3Myc complexes, aliquots of 2 × 10⁸ cells were harvested and lysed in 500 μL of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl containing different detergents; 0.5% digitonin, 1% digitonin, 1% Triton X-100 (TX-100), 1% n-octyl-beta-glucoside (NOG) or 1% decyl-β-D-maltopyranoside (DM). After centrifugation at 16,000 g, 4°C for 20 min, aliquots of the supernatants equivalent to 2 x 10⁸ cells were incubated with 10 μL anti-Myc agarose beads (Myc-Trap^TM, Chromotek) for 1 h at 4°C. The beads were washed three times in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl containing the corresponding detergents and bound proteins were eluted three times with 10 μL 0.5 mg/mL c-Myc peptide (Sigma M2435) in the corresponding detergent containing buffer. The combining eluates for each detergent condition, equivalent to 2 × 10⁸ cells, were subjected to NativePAGE (Invitrogen) and transferred to a PVDF membrane (Invitrogen) followed by immunoblotting with anti-Myc antibody (Chromotek, 9E1) diluted 1:1,000. The blot was then developed by ECL using an HRP-conjugated secondary antibody (Sigma, A9037, 1:3,000).

For exogenous Co-IP analysis, plasmids expressing PTPN2 and other indicators were transfected into HEK293T cells. After transfection with 24 h, cells were lysed with 1 mL Cell Lysis for protein extraction. After centrifugation for 15 min at 13,000g, 4°C, 100ul supernatant was removed as “Input” and the rest was incubated with agarose immunoprecipitation beads for tag combination with 4 °C incubation overnight on a rotary mixer. Beads were then washed for 5 times using Radio Immunoprecipitation Assay (RIPA) buffer (P0013c, Beyotime Biotechnology, China). Precipitated proteins were separated from beads after incubation at 99°C for 5min with 2x loading buffer. Before the incubation at 99°C, c-Myc peptide (M2435, sigma) or 3X-Flag peptide (F4799, sigma) was added to compete out the binding of c-Myc or 3X-Flag for necessary.
The protocol of endogenous Co-IP in 3T3-L1-derived adipocytes was similar to exogenous analysis described above. After supernatant preparation, corresponding antibodies were added for endogenous protein combination at 4°C for 1 h. Then protein A/G Plus Agarose (sc-2003, Santa Cruz Biotechnology, United States) was used for antibody-combination at 4°C overnight with gentle mixture. Notably, normal rabbit IgG (2729S, Cell Signaling Technology, United States) was used as a negative control (Ida et al., 2021 (link)).