Journal: Journal of Biological Chemistry

Article Title: Heat Shock Protein 27 Functions in Inflammatory Gene Expression and Transforming Growth Factor-β-activated Kinase-1 (TAK1)-mediated Signaling

doi: 10.1074/jbc.m610987200

Figure Lengend Snippet: FIGURE 4. HSP27 regulates IL-1-induced p38 MAPK and JNK activity but not ERK activation. Cells were transfected as indicated and treated with IL-1 for different times. Cells were harvested and lysed. A, p38 MAPK was immunoprecipitated from lysates and assayed for its ability to phosphorylate His-tagged MK2. Graph shows mean p38 MAPK activity from three independent experiments (S.E.) as a percentage of peak activity in Scr-transfected cells. A portion of the lysates was Western-blotted for HSP27 and p38 MAPK. B, JNK was immu- noprecipitated from lysates, and JNK activity was assayed with GST-ATF-2 as a substrate. Graph shows JNK activity (as for A). An aliquot of lysate was analyzed by Western blotting for HSP27 and -tubulin. C, activation of ERK in lysates was detected by Western blotting with an antibody that recognizes ERK phos- phorylated on Thr-185 and Tyr-187. The blot was stripped and re-probed for total ERK. D, cells were transfected with Scr, HSP27#2, or HSP27#5 twice (with a 24-h interval between transfections) and harvested and lysed 24 h after the second transfection. Phosphorylation of p38 MAPK on Thr-180 and Tyr-182, JNK1 on Thr-183 and Tyr-185, JNK2 on Thr-221 and Tyr-223, and ERK (as in C) in lysates was examined by Western blotting with phosphospecific antibodies. p38 MAPK was blotted as a loading control. Data in each panel (A–D) are repre- sentative of at least two independent experiments.

Article Snippet: The membranes were stained with rabbit anti-COX-2 antibody (Alexis), a mouse anti- -tubulin antibody DM-1A (Sigma-Aldrich), a mouse monoclonal antibody against actin (Sigma), a rabbit antiserum raised against MKK3/6 (32), a rabbit antiserum to human HSP27 (7), a rabbit antiserum that recognizes both human HSP27 and murine HSP25 (Stressgen), a rabbit antiserum to the C-terminal peptide of p38 MAPK (33), a rabbit antiserum against TAK1 (sc-7162; Santa Cruz Biotechnology), and a rabbit antiserum against I B (sc-203; Santa Cruz Biotechnology).

Techniques: Activity Assay, Activation Assay, Transfection, Immunoprecipitation, Western Blot, Phospho-proteomics, Control

Journal: Journal of Biological Chemistry

Article Title: Heat Shock Protein 27 Functions in Inflammatory Gene Expression and Transforming Growth Factor-β-activated Kinase-1 (TAK1)-mediated Signaling

doi: 10.1074/jbc.m610987200

Figure Lengend Snippet: FIGURE 5. HSP27 is needed for activation by IL-1 of the p38 MAPK activators (MKK3 and MKK6) and the JNK activators (MKK4 and MKK7). Cells were transfected and treated as before. A, activation of MKK3 and MKK6 was detected with an antibody that recognizes phosphorylation of Ser-189 and Thr-193 of MKK3 and Ser-207 and Thr-211 of MKK6. The blot was stripped and re-probed with an antibody that recognizes total MKK3 and MKK6. B, kinase assay for MKK4 using a GST-JNK2 (K55R) (a mutant form that is incapable of auto- phosphorylation) and Western blots for HSP27 and -tubulin. C, kinase assay for MKK7 (as for B). Similar results were obtained in two separate experiments for each panel.

Article Snippet: The membranes were stained with rabbit anti-COX-2 antibody (Alexis), a mouse anti- -tubulin antibody DM-1A (Sigma-Aldrich), a mouse monoclonal antibody against actin (Sigma), a rabbit antiserum raised against MKK3/6 (32), a rabbit antiserum to human HSP27 (7), a rabbit antiserum that recognizes both human HSP27 and murine HSP25 (Stressgen), a rabbit antiserum to the C-terminal peptide of p38 MAPK (33), a rabbit antiserum against TAK1 (sc-7162; Santa Cruz Biotechnology), and a rabbit antiserum against I B (sc-203; Santa Cruz Biotechnology).

Techniques: Activation Assay, Transfection, Phospho-proteomics, Kinase Assay, Mutagenesis, Western Blot

Journal: British journal of cancer

Article Title: Inactivation of the WASF3 gene in prostate cancer cells leads to suppression of tumorigenicity and metastases.

doi: 10.1038/sj.bjc.6605850

Figure Lengend Snippet: Figure 7 Knockdown of WASF3 expression suppresses MMP9 expression levels and inhibits MMP9 release from prostate cancer cell lines. (A) RT–PCR analysis of PC3 and DU145 cells showing knockdown of WASF3 also show down-regulation of MMP9, but not MMP2 expression levels. Cont ¼ parental cells. (B) Secretion of the pro- and active form of MMP9 from PC3 and DU145 cells were measured in the supernatants of actively growing cells using the Fluorokine MAP assay procedure. Cells from each clone showing knockdown of WASF3 also showed reduced MMP9 levels compared with parental (control) and non-knockdown cells that did not.

Article Snippet: After 24 h, the media were collected in tubes and centrifuged for 10 min at 10 000 g. The pro- and active MMP-9 levels released into the media were measured using a Fluorokine MAP human MMP9 kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Control