Rabbit α hsp27 phospho ser82

2 mL of MCF10A cell suspension at a cell concentration of 1.5 × 10⁵ cells/ml was seeded into a 6-well plate (Falcon, #353046) and grown for 48 h at 37°C. The medium was exchanged and the cells were grown for another 6 h. An equal volume of medium containing the prediluted inhibitors was then added to the cells, and the cells were incubated for 30 min (for PD0166285) or 6 h (for cycloheximide and cycloheximide plus either SB202190 or SB203580). The medium was then removed, cells were washed twice with cold PBS and lysed with lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1x Phosstop #4906845001, 1x cOmplete #11873580001). Samples were boiled in SDS-PAGE sample buffer and analyzed by SDS-PAGE and immunoblotting. The following antibodies were used: rabbit α-Cdk1 phospho-Tyr15 (Cell Signaling Technology, #9111L), mouse α-Cdk1 (Santa Cruz Biotechnology, #SC-54), mouse α-tubulin (Santa Cruz Biotechnology, #SC-32293), rabbit α-HSP27 phospho-Ser82 (Cell Signaling Technology, #2401), mouse α-HSP27 (Cell Signaling Technology, #2402), rabbit α-p38 MAPK (Cell Signaling Technology, #9212) and rabbit α-p38 MAPK phospho-Thr180/Tyr182 (Cell Signaling Technology, #9211).