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Published byIrwan Sumadi Modified over 5 years ago
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pHSF1Ser230 is upregulated by TNF in colon cancer cells.
pHSF1Ser230 is upregulated by TNF in colon cancer cells. (A) Peptide alignment of HSF1 from various species. Putative DAPK phosphorylation sites are boxed in gray. (B) HCT116 cells (DAPK wt) and HCT116 cells with DAPK knocked down by shRNA (shDAPK) were treated with 0.66 ng/ml TNF. At the indicated time-points, whole-cell lysates were prepared and equal amounts of protein were resolved by SDS-PAGE and probed with the specific antibodies indicated. GAPDH was used as a loading control. Ctrl, control. (C) Colorectal tumor tissue lysates were immunoblotted with antibodies as indicated. γ-tubulin was used as a loading control. (D) Interaction of the DAPK–HSF1–CaMKII complex. The catalytic domain of DAPK (PDB ID: 1JKS) was docked to HSF1 modeled using the PDB IDs 2LDU (10–123), 4DZM and 1EXU (126–203), 3K9J (200–260), 2VBC (203–310), 1Z05 (311–371), 2WLX and 3OOQ (371–529). CaMKII in its active form was modeled with the PDB ID 3KK8 as a template and was then docked to the DAPK–pHSF1Ser230 complex. HSF1 is shown in green, CaMKII is indicated in blue and the DAPK kinase domain is shown in pink. Phosphorylated residue Ser230 of HSF1 is shown as a red sphere. (E) HCT116 cells were treated with 10 µM KN62 (CaMKII inhibitor) or 10 µM DAPK inhibitor (DI), with or without 0.66 ng/ml TNF. At 24 h after treatment, cell lysates were resolved by SDS-PAGE and probed with the specific antibodies indicated. GAPDH was used as a loading control. (F) Recombinant HSF1–His (1 µg) was incubated with recombinant CaMKII (0.5 µg) and ATP (200 µM) in CaMKII kinase buffer in the presence of 10 µM KN62 (CaMKII inhibitor) or 10 µM DAPK inhibitor at 30°C for 30 min. Proteins were resolved by SDS-PAGE. The level of phosphorylation was determined using a specific antibody against pHSF1Ser230. (G) Colorectal adenocarcinoma cell lines DLD1 and Caco2 were treated with 0.66 ng/ml TNF. At the indicated time-points, cell lysates were resolved by SDS-PAGE and probed with the specific antibodies indicated. GAPDH was used as a loading control. (H) Western blot analysis of HCEC cell lysate treated with 0.66 ng/ml TNF for the indicated times. Blots were immunoprobed with the indicated antibodies. Blots from representative experiments are shown (n = 3). In all cases the ratio of pHSF1Ser230∶HSF1 was calculated using ImageJ and the control of each cell line was adjusted to 1. Natalya Benderska et al. J Cell Sci 2014;127: © Published by The Company of Biologists Ltd
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