HeLa cells were lysed in the presence of 0.1% Triton X-100 and the 10,000×g soluble supernatants were applied to sepharose CL-6B gel filtration columns as described in Materials and methods.
29, 66, 150, 200, 443, 669 and 2000 (kDa) indicate the apparent native size of gel filtration markers. A, The presence of Hsp27, HDAC6, STAT2 and procaspase-3 in pooled fractions eluted from the columns was detected by western blot analysis. B, interactions between Hsp27 and client proteins analyzed by Co-IP experiments using a goat polyclonal anti-Hsp27 antibody. Column fractions corresponding to either procaspase-3, STAT2 or HDAC6 were pooled and used for immunoprecipitation. Immunoprecipitated proteins and input cell lysates were analyzed side by side in immunoblots probed with the indicated antibodies. A representative negative control of Co-IP was performed with a control antibody and revealed (here the anti procaspase 3).