Western blot analysis

Immunoblotting was carried out with 50 μg protein on 12.5% SDS-PAGE. The electrophoresis was performed at room temperature and the proteins were electroblotted onto Hybond-C membrane (Amersham Biosciences, U.K.) at 150 mA for 2 h. The membranes were blocked with 5% (w/v) nonfat milk in TBST buffer (0.1 M Tris pH 7.9, 0.15 M NaCl and 0.1% Tween 20) and probed with primary polyclonal anti-MSR (ab16803) and anti-DnaK (ab80161) (Abcam Ltd., U.K.) at varying dilutions (1:1000–1:15000) in TBS buffer. Immunodetection was performed with horse radish peroxidase conjugated anti-goat IgG (Abcam Ltd., U.K.) as secondary antibody at a concentration of 1:20000 for detection using enhanced chemiluminescence (Pierce). X-ray film from immunoblotting was scanned using Bio-Rad FlourS system equipped with a 12-bit camera and exported in tiff format. Protein quantification was performed using the Fluor S MultiImager (Bio-Rad) and Quantity 1-D Analysis software (Bio-Rad) using the volume analysis function, and the relative signals were calculated.