Cells derived from our experimental conditions were lysed in radioimmunoprecipitation assay (RIPA) buffer and 30 μg of total proteins were loaded on precast SDS-PAGE gels (BioRad), separated and transferred onto PVDF membrane. Immunoblots were incubated overnight in 5% BSA or milk in T-PBS at 4°C and probed with primary and appropriate secondary Horseradish Peroxidase (HRP)-antibodies. Immunoprecipitation was performed on 500 mg of total proteins in 500 ml of RIPA buffer using 20 μl of protein A/G plus (Santa Cruz Biotechnology) per samples, following manufacturer's instruction.
Detection of PKM2 redox state, N-(biotinoyl)-N’-(iodoacetyl) ethylene diamine BIAM labeling was performed as previously described [34 (link)]. Briefly, cells were rapidly rinsed in liquid nitrogen and exposed to RIPA buffer containing 100 μM of BIAM diamine and incubated for 15 min at room temperature. Lysates were then clarified by centrifugation and 500 μg of each sample were suspended into 500 μl of RIPA buffer and immunoprecipitated using 20 μl of Streptavidin-agarose beads (Sigma). Immunocomplexes labelled with BIAM were separated by SDS–PAGE and the biotinylated/reduced fraction was revealed with HRP-conjugated.
Detection of PKM2 redox state, N-(biotinoyl)-N’-(iodoacetyl) ethylene diamine BIAM labeling was performed as previously described [34 (link)]. Briefly, cells were rapidly rinsed in liquid nitrogen and exposed to RIPA buffer containing 100 μM of BIAM diamine and incubated for 15 min at room temperature. Lysates were then clarified by centrifugation and 500 μg of each sample were suspended into 500 μl of RIPA buffer and immunoprecipitated using 20 μl of Streptavidin-agarose beads (Sigma). Immunocomplexes labelled with BIAM were separated by SDS–PAGE and the biotinylated/reduced fraction was revealed with HRP-conjugated.
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