Journal: Genes & Development

Article Title: ATR checkpoint kinase and CRL1 βTRCP collaborate to degrade ASF1a and thus repress genes overlapping with clusters of stalled replication forks

doi: 10.1101/gad.239194.114

Figure Lengend Snippet: DOX induces ATR-CRL1 βTRCP -dependent ASF1a degradation. ( A ) Immunoblots of cell lysates. ASF1a, but not ASF1b or CAF1, was decreased by DOX for 20 h, and this decrease was relieved by 5 mM caffeine added to cells from −4 h relative to DOX until the time of harvest. ACTIN was used as a loading control for the immunoblots. ( B , left panel) ASF1a/b examined in cells transfected with siGL2, siATR, or siATM for 24 h and then treated with DOX for 20 h under continued exposure to siRNAs. ( Right panel) Knockdown of endogenous ATR or ATM by siRNA was confirmed by immunoprecipitation (IP) and Western blot (WB). The IgG amount in each lane served as the loading control for the immunoprecipitates, and ACTIN shows equal amounts of whole -cell extracts (WCEs). ( C ) Cells treated with DOX for 20 h were exposed to 10 μM MG132 for 4 h before harvest. Endogenous ASF1a and ACTIN were immunoblotted. ( D ) Cells transfected with two different siRNAs to βTRCP for 24 h and a siRNA to Cullin1 for 48 h before DOX treatment for 20 h with continued incubation with siRNA and subjected to Western blotting for the indicated proteins and ACTIN (loading control). ( E ) After transfection of siGL2 or siATR (24 h) or exposure to 5 mM caffeine (4 h), cells were treated with DOX or DMSO for 20 h with continued incubation with siRNA or caffeine. Cells were harvested after exposure to 10 μM MG132 for 4 h to stabilize the ASF1a. Immunoprecipitates ( top four panels) or input whole-cell extracts ( bottom three panels) were immunoblotted for the indicated proteins. IgG served as a loading control for immunoprecipitates, and Cullin1 served as a loading control for whole-cell extracts. ( F ) An experiment similar to E , except immunoprecipitation done with anti-βTRCP antibody.

Article Snippet: Antibodies against each protein were purchased from the following companies: CHK1, Cyclin A, Cyclin E, CUL1, and CAF1 from Santa Cruz Biotechnology; p-CHK1(Ser345), ASF1a, ASF1b, and βTRCP from Cell Signaling Technology; ATR from Affinity Bioreagent; ATM from Novus Biological; and BRCA1 from Milipore.

Techniques: Western Blot, Control, Transfection, Knockdown, Immunoprecipitation, Incubation

Journal: Genes & Development

Article Title: ATR checkpoint kinase and CRL1 βTRCP collaborate to degrade ASF1a and thus repress genes overlapping with clusters of stalled replication forks

doi: 10.1101/gad.239194.114

Figure Lengend Snippet: The ATR-dependent checkpoint pathway is required for the transcriptional repression of genes overlapping with clusters of stalled forks. ( A , B ) Histone H3 ( A ) and RPA ( B ) ChIP assay for three genes overlapping with stalled forks and a control distal gene, WNT2 . The rest are as in . (DOX-CF) DOX-treated cells exposed to caffeine for 24 h before harvesting. ( C ) RNA polymerase II (pol II) ChIP assay at the promoters of three genes overlapping with stalled forks and a control distal gene, WNT2 . (DOX-CF) DOX-treated cells exposed to caffeine for 24 h before harvesting. ( D ) Cells depleted of ATR or ATM by siRNA were treated with 1.5 μM DOX or DMSO (Con). The relative mRNA expression of three overlapped genes and a control gene, p21 , was analyzed by qRT–PCR as in the Materials and Methods. Student’s t -test analysis, (*) P < 0.05.

Article Snippet: Antibodies against each protein were purchased from the following companies: CHK1, Cyclin A, Cyclin E, CUL1, and CAF1 from Santa Cruz Biotechnology; p-CHK1(Ser345), ASF1a, ASF1b, and βTRCP from Cell Signaling Technology; ATR from Affinity Bioreagent; ATM from Novus Biological; and BRCA1 from Milipore.

Techniques: Control, Expressing, Quantitative RT-PCR