Figure 4
Effect of heregulin α1 (HRG α1) and p38 MAPK inhibitor (SB 203580) on Hsp27 phosphorylation. Cultures of cells from the BT474 breast cancer cell line were treated with HRG α1 for 10 and 30 min (A) or SB 203580 for 10 hours (B) and total cell lysates were extracted using M-PER reagent (Pierce). Equal amounts of proteins (20 μg) were separated by SDS-PAGE and transferred onto PVDF membrane. The membranes were blocked for 1 hour, followed by being probed with the respective primary antibodies (anti-Hsp27, anti-pSer15, anti-pSer78 and anti-pSer82), and HRP-conjugated secondary antibody. The signals were captured and their intensities were detected as described in Figure 2. The phosphorylation levels of pSer15, pSer78 and pSer82 were expressed as the ratios of intensity probed with phosphorylation site-specific antibody to the intensity probed with anti-Hsp27. Data with ± SD represents the average of triplicate experiments. C: control; SB: inhibitor SB203580. For the control of HRG-treated cells, untreated cells were cultured for 10 and 30 min and equal amounts of cellular proteins from both time intervals were mixed and used as control. For the control of inhibitor-treated cells, cells were treated with DMSO for 10 hours and cellular proteins were used as the control. *p < 0.05 (student t-test)