Hrp conjugated goat secondary antibodies

For immunoblotting, cells were trypsinized and washed with ice-cold PBS, lysed in lysis buffer (50 mM Tris-HCl PH 8.0, 1% SDS, 1 mM EDTA, 5 mM DTT, 10 mM PMSF, 1 mM NaF, 1 mM Na₃VO₄, and protease inhibitor cocktail), and then denatured in boiling water for 10 min. The cellular lysates were centrifuged (13,000 × g, 30 min), and protein concentration was determined using a BCA assay (ThermoFisher, 23225). Cell lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked with 5% skim milk and incubated with the indicated antibodies at 4 °C overnight. HRP-conjugated goat secondary antibodies were used (1:5000, Invitrogen). Then membranes were washed three times with TBST followed by secondary antibody incubation for 2 h at room temperature. Immunodetection was achieved with the chemiluminescence reagent (Thermo) and detected by a GE ECL machine.

For immunoblotting, polypeptides were resolved by SDS–PAGE and transferred to a PVDF membrane (Bio-Rad). Membranes were blocked with 5% non-fat dry milk, and probed with the indicated antibodies. HRP-conjugated goat secondary antibodies were used (1:10,000, Invitrogen). Immunodetection was achieved with the Hyglo chemiluminescence reagent (Denville Scientific), and detected by a Fuji ECL machine (LAS-3000). For co-immunoprecipitation, cells were lysed in 1% NP40 lysis buffer (25 mM Tris pH 7.5; 300 mM NaCl, 1 mM EDTA, 1% NP40), supplemented with a complete protease inhibitor cocktail (Roche). After preclearing with protein A/G agarose beads for 1 hr at 4 °C, whole-cell lysates were used for immunoprecipitation with the indicated antibodies. Generally, 1–4 μg commercial antibody was added to cell lysate, which was incubated at 4 °C for 8–12 h. After addition of protein A/G agarose beads, incubation was continued for another 2 h. Immunoprecipitates were extensively washed with NP40 lysis buffer and eluted with SDS–PAGE loading buffer by boiling for 5 min before resolution by SDS–PAGE.