Quantitative Western Blot Analysis

For protein extraction, exponentially growing cells of the strains NKY2 and NKY43 were taken. To achieve the functional loss of Hsp82 in NKY45 strains, one batch of cells was grown at 37°C overnight, and the other batch of cells was allowed to grow at 25°C. Equal amounts of cells were finally harvested, and protein was isolated from them by the trichloroacetic acid (TCA) method and subsequently followed for Western blotting (39 (link)). The antibodies used were mouse anti-Act1 antibody (Abcam) and mouse anti-Hsp82 antibody (Calbiochem) at 1:5,000 dilutions. Rabbit anti-Myc antibody (Abcam) was used at 1:8,000 dilutions. For secondary antibodies, horseradish peroxide-conjugated anti-rabbit antibody (Promega) and anti-mouse antibody (Santa Cruz Biotechnology Inc., CA, USA) were used at 1:10,000 dilutions. The Western blots were developed using a chemiluminescent detection system (Pierce). The bands on the blots were quantified using ImageJ software, and the relative densities thus obtained were plotted using GraphPad Prism 6 software. The mean values from three independent experiments were plotted (± standard deviation [SD]).