Anti phospho hsp27

The following reagents and materials were used: anti-GAPDH (Calbiochem), anti-MKK3, anti-MKK6, anti-phospho MKK3 (Ser189)/MKK6 (Ser207), anti-p38 MAP kinase, anti-phospho p38 MAP kinase (Thr180/Tyr 182), anti-phospho HSP27 (Ser82), anti-phospho ATF2 (Thr71), anti-PDK1, anti-phospho PDK1 (S241), anti-PKC-Pan, anti-phospho PKC-pan (T514), anti-Prohibitin 1 (Phb1), anti-Prohibitin 2 (Phb2), (Cell signaling). Anti-vimentin antibody was purchased from Santa Cruz biotechnology. Anti-phospho Phb1 (T258), and anti-phospho Phb1 (Y259) were purchased from Signalway Antibody. Electrophoresis reagents were purchased from Biorad and trypsin from Promega.

The following antibodies were used for Western blot analyses, immunoprecipitation, and immunohistochemistry: anti-mTOR, anti-phospho-mTOR^Thr2448, anti-p70S6K, anti-phospho-p70S6K^T389, anti-4EBP1, anti-phospho-4EBP1^Thr37/46, anti-phospho-4EBP1^Ser65 and anti-phospho-HSP27 from Cell Signaling; anti-PRAK, anti-HIF-1α and anti-MMP2 from Proteintech (Chicago, IL), anti-tubulin and anti-GAPDH from Ruiying Biological (Suzhou, China), anti-vimentin from Gene Tex (San Antonio, Texas), anti-Snail1, anti-MMP9, anti-CDH1(E-cadherin) and anti-CDH2 (N-cadherin) from ABclonal (Wuhan, China), anti-Twist1 from Signalway Antibody (College Park, Maryland), Isotype control IgG from Sigma. PRAK inhibitor GLPG0259 was synthesized by Wuxi Pharma (Shanghai, China). MK2 inhibitor PF 3644022 was purchased from R&D systems. mTORC1 inhibitor rapamycin and mTORC1 agonist MHY1485 were from Selleck (Houston, TX), Texas red-labeled tomato lectin was from Vector Laboratories (Burlingame, CA).

RPMI8226 cells were seeded and treated with either 500 nM of test compounds or 0.1 % DMSO. After incubation for 1 h, cells were washed with media three times, and incubated for an additional 4 or 24 h. Cells were washed with ice-cold PBS and lysed with Pro-Prep lysis buffer (iNtRON Biotechnology). Protein lysates were analyzed by Western immunoblotting using the following antibodies: anti-Ubiquitin (Enzo Life Sciences), anti-Hsp70 and anti-Hsp27 (Abcam), anti-Gadd34 (Proteintech), anti-caspase-8 (BD pharmingen), anti-β-Catenin, anti-p21, anti-phospho Hsp27 (S82), anti-caspase-9, anti-PARP, and anti-α-Tubulin (Cell Signaling). Images were obtained using ChemiDoc MP (Bio-Rad) and quantitation was done using Image Lab Software (Bio-Rad).