Briefly, plasmids expressing the replicon SinRep EGFP or DHBB helper RNAs had been linearized with PacI or XhoI respectively. In vitro transcription was performed applying the mMessage mMachine RNA transcription kit . Helper and replicon RNAs have been then electroporated into BHK cells and incubated in MEM supplemented with FCS for h. Immediately after h, the media was replaced with OPTI MEM supplemented with mg l CaCl and cells had been incubated at ?C for h, at which time the supernatant was collected, spun at g, ?C to remove debris, and frozen at ? ?C. Vectors had been titered as previously described . Repication competent virus, carrying the luciferase gene, was also created from DNA plasmids . Cells have been contaminated with SV EGFP in OPTI MEM CaCl at a multiplicity of infection of , to realize greater than infectivity as assessed by fluorescent microscopy. Mock contaminated cells have been incubated in OPTI MEM CaCl. Cultures have been gently rocked at ?C for h just before elimination of virus or media, washed with PBS then incubated in total media at ?C for indicated times; time submit infection was calculated through the time at ?C incubation Western blotting Cell lysates were ready utilizing Total Cell Lysis Buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor .
Cells were harvested, washed when in PBS, then rotated at ?C for min before spinning at , g at ?C for min to take away debris. Protein concentrations have been measured applying BioRad Dc Protein Reagent. Protein samples were run on gradient SDS polyacrylamide gels beneath reducing ailments. Protein was transferred to polyvinylidene fluoride membrane in Tris glycine buffer pH . containing methanol. Antibodies utilized: anti ATM phospho Ser , anti Mcm , anti Chk and actin , anti p phospho Ser purmorphamine , anti phospho HA.X Ser and anti Chk antibodies. Horseradish peroxidase conjugated secondary antibodies were utilized and filters developed with SuperSignal West Pico Chemiluminescence substrate and exposed to autoradiography film . Densitometry of scanned autoradiographs was carried out by using NIH Image J.f software Immunoprecipitation Dynal beads were incubated with g anti ATM phospho Ser or anti Mcm for h at RT with rotation followed by two washes with PBS Tween .
Beads have been then rotated with g lysate overnight at ?C. Samples had been washed with PBST. Protein was eluted from beads with l SDS Page sample buffer. commercial compound libraries For mass spectrometry analysis, g full cell lysate was primary pre cleared with mouse IgG bound beads prior to overnight incubation with anti phospho ATM. Beads had been washed with PBST M KCl, then with PBST Mass spectrometry Cell lysate prepared just after h SV EGFP infection was immunoprecipitated and run on the SDS Webpage gel. The gel was visualized with Coomassie Blue stain. Protein isolation, digestion and examination by MALDI TOF have been carried out through the Rockefeller University Proteomics Facility .