Journal: Nature Communications
Article Title: BRE/BRCC45 regulates CDC25A stability by recruiting USP7 in response to DNA damage
Figure Lengend Snippet: BRE overexpression affects IR-induced CDC25A degradation. a DNA synthesis in ES cells after different doses of IR. b Western blot analysis of CDC25A and Tyr15 phosphorylation of CDK1 (p-CDK1) at different times after exposing the cells to 6 Gy IR in Brca2 CKO/KO ;MSCV-BRE cells compared to Brca2 CKO/KO cells. GAPDH was used as a loading control. Numbers below indicate the lane numbers. The quantification of CDC25A (middle panel) and p-CDK1 (lower panel) band intensity for each cell line is shown as histogram. Relative band intensities were calculated by dividing GAPDH-normalized CDC25A/p-CDK1 intensity at particular time point with GAPDH-normalized CDC25A/p-CDK1 intensity of corresponding untreated cells. c MCF7 cells with inducible HA-BRE expression cassette were transfected with CRE plasmid and subjected to 6 Gy IR after 48 h of transfection. Western blots of endogenous CDC25A in presence (lanes 4–6) and absence (lanes 1–3) of HA-BRE expression at different times after irradiation is shown in the upper panel. Lower panel shows the quantification of CDC25A band intensity normalized against GAPDH. d Immunoblot showing CDC25A degradation after IR treatment in MCF7 cells transfected either with non-specific (NS) siRNA or two different siRNAs against BRE (#1 and #2). Histone H3 was used as a loading control. Relative CDC25A band intensities were calculated by dividing GAPDH-normalized CDC25A intensity at a particular time point with GAPDH-normalized CDC25A intensity of untreated (0 h IR) cells of corresponding siRNA treatment. e Western blot showing IR-induced CDC25A degradation in MCF7 cells with stably integrated CRE inducible HA-BRE expression cassette after transfection either with non-specific (NS) siRNA or two different siRNAs against MERIT40 (#1 and #2) along with CRE -expressing plasmid. Non-specific (NS) siRNA transfection was used as control. Relative abundance of CDC25A at a particular time point was measured by comparing GAPDH-normalized band intensities at that point divided by GAPDH-normalized CDC25A intensity of untreated (0 h IR) cells of corresponding treatment. All histograms show the average of three independent experiments and error bars represent s.d. P values were calculated using paired two-tailed t
Article Snippet: The antibodies used for co-immunoprecipitation, immunofluorescence, and western blots were anti-BRE (C-16: sc-48847 and sc-376453; Santa Cruz Biotech, 1:500 dilution for western blots), anti-BRCC36 (ab62075; Abcam and sc131122; Santa Cruz Biotech, 1:1000 dilution for western blots), anti-CDC25A (F-6: sc-7389; Santa Cruz Biotech, 1:200 dilution), anti-FLAG (F7425 and F3165; Sigma-Aldrich, 1:2000 dilution for western blot), anti-HA (12013819001; Roche, 1:2000 dilution), anti-USP7 (H-200: sc-30164 and H12: sc-137008; Santa Cruz Biotech and ab4080; Abcam, 1:1000 dilution for western blot), anti-β TRCP (ab118006; Abcam, 1:500 dilution), anti-DUB3 (ab174914; Abcam and H00377630-M01; Abnova, 1:1000 dilution for western blot), anti-RAP80 (A300-764A-M; Bethyl laboratories, 1:500 dilution for western blot), K-48 linkage specific antibody (#4289S; Cell Signaling, 1:500 dilution), K-63 linkage specific antibody (#5621S; Cell Signaling, 1:500 dilution), anti-histone H3 (#9715; Cell Signaling, 1:5000 dilution), anti-HDM2 (sc-56154; Santa Cruz Biotech, 1:500 dilution), anti GAPDH (2118S; Cell Signaling, 1:40,000 dilution), anti-ACTIN (C2: sc-8432; Santa Cruz Biotech, 1:40,000 dilution), anti-phosphorylated CHK1 (12302; Cell Signaling, 1:500 dilution), anti-phosphorylated CDK1 (#9111; Cell Signaling, 1:1000 dilution), anti-p53 (#2524; Cell Signaling, 1:5000 dilution), anti-RAD51 (PC130; Calbiochem, 1:2000 for western and 1:250 for Immunofluorescence), anti-γH2AX (05-636; Upstate), anti-ubiquitin (P4D1: sc-8017; Santa Cruz Biotech, 1:1000 dilution), anti-Myc (631206, Clontech and 2272S; Cell Signaling, 1:1000 dilution), anti-MERIT40 (#12711; Cell Signaling, 1:1000 dilution), and anti-BRCA2 (A303-434A-T; Bethyl Laboratories, 1:2000 dilution).
Techniques: Over Expression, DNA Synthesis, Western Blot, Expressing, Transfection, Plasmid Preparation, Irradiation, Stable Transfection, Two Tailed Test