Exogenous and Endogenous Co-Immunoprecipitation Analysis

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)).