Journal: Cellular and Molecular Life Sciences
Article Title: Essential role of CK2α for the interaction and stability of replication fork factors during DNA synthesis and activation of the S-phase checkpoint
Figure Lengend Snippet: Down-regulation of CK2α affects the expression of cell cycle regulatory proteins implicated in the response to DNA replication stress. A Scheme summarizing the eukaryotic replisome complex coordinating DNA replication. DNA replication is carried out by specific DNA polymerases (Pol) on the leading and legging strands, respectively. Stretches of ssDNA are coated and stabilized by replication protein A (RPA). Unwinding of the DNA strands is carried out by minichromosome maintenance protein complex (MCM2-7) associated with cell division cycle 45 (CDC45) and go-ichi-ni-san (GINS) proteins. Altogether, they form the CMG complex. DNA replication is a process carried out with high fidelity. In order to accomplish this, eukaryotic cells express checkpoint proteins whose task is to closely monitor the genome and detect errors or problems during DNA replication. Checkpoint proteins are evolutionary well conserved and include ATR, ATRIP, CHK1, CLSPN, CDK2, Cdc7 which, altogether, mainly respond to stretches of ssDNA. Rad9–Hus1–Rad1 (9–1–1) is a heterotrimeric clamp on DNA sufficient to facilitate the interaction between ATR, its partner ATRIP, TOBP1 and CLSPN. During activation of a stress response following perturbation of DNA replication, CLSPN docks with CHK1 the major checkpoint effector kinase, contributing to promote the activation of surveillance mechanisms at the replisome. B , C Cells were incubated with vehicle or 1 μg/ml Dox for 72 h and then, where indicated, added 3 mM HU for 4 h ( B ), 6 h or 24 h ( C ). Harvested cells were processed as described in the materials and methods and whole cell lysates were analysed probing Western blot membranes with antibodies directed against proteins indicated in the figure. Experiments were performed at least three times obtaining similar results. β-Actin detection was used as loading control. One representative experiment is shown. Exp: exposure. Where indicated, the intensity of protein bands was quantified by densitometric analysis using ImageJ software (NIH) and calculated in percentage by assigning a value of 100% to specific bands as indicated in the figure
Article Snippet: Proteins were detected by incubating PVDF membranes with the following primary antibodies: rabbit polyclonal anti-phospho-NF-κB S529 (ab47395, Abcam, Cambridge, United Kingdom), rabbit monoclonal anti-phospho-CHK1 S345 (2348, Cell Signaling Technology), mouse monoclonal anti-CHK1 (2360, Cell Signaling Technology), rabbit monoclonal anti-cyclin E1 (20808, Cell Signaling Technology), mouse monoclonal anti-p53 (2524, Cell Signaling Technology), rabbit polyclonal anti-phospho-p53 S15 (9284, Cell Signaling Technology), rabbit polyclonal anti-Cdc7 (3603, Cell Signaling Technology), rabbit monoclonal anti-NF-κB (8242, Cell Signaling Technology), rabbit monoclonal anti-CDC45 (11881, Cell Signaling Technology), rabbit monoclonal anti-phospho-histone H2A.X S139 (9718, Cell Signaling Technology), mouse monoclonal anti-α-Tubulin (3873, Cell Signaling Technology), rabbit monoclonal anti-histone H3 (4499, Cell Signaling Technology); rabbit polyclonal anti-cyclin A (sc-751, Santa Cruz Biotechnology, Heidelberg, Germany), mouse monoclonal anti-ATR (sc-515173, Santa Cruz Biotechnology), mouse monoclonal anti-ATRIP (sc-365383, Santa Cruz Biotechnology), mouse monoclonal anti-MCM7 (sc-9966, Santa Cruz Biotechnology), mouse monoclonal anti-CLSPN (sc-376773, Santa Cruz Biotechnology), goat polyclonal anti-MCM3, mouse monoclonal anti-CDK2 (sc-6248, Santa Cruz Biotechnology), mouse monoclonal anti-HDAC2 (sc-9959, Santa Cruz Biotechnology), and mouse monoclonal anti-β-actin (A-5441, Sigma-Aldrich).
Techniques: Expressing, Activation Assay, Incubation, Western Blot, Software