Co-immunoprecipitation (Co-IP) assay

Equal amounts of cell lysates (1500 μg) were subjected to immunoprecipitation (IP) overnight using 1 μg of specific antibodies, such as HSP70, along with 100 μL of protein A/G agarose with continuous rotation. The immunoprecipitants were washed twice with 1 mL 10 mM HEPES (pH 7.9), 1 mM EDTA, 150 mM NaCl, and 1% Nonidet P-40. The precipitated proteins were eluted with 60 μL SDS-PAGE sample buffer by boiling for 10 min. The eluted proteins were separated on a 6% SDS-PAGE gel, transferred to nitrocellulose membranes, and incubated with the specified antibodies.