Goat anti mcm2

Free floating tissue sections were triple washed in PBS, then incubated for 1 hour in blocking solution (12.5% normal donkey serum and 0.25% triton in PBS), followed by incubation with one or more of the following primary antibodies diluted in blocking solution: goat anti-MCM2 1:100 (Santa Cruz Biotechnology, Cat# sc-9839), mouse anti-GFAP 1:500 (Millipore, Cat# MAB3402), rabbit anti-Kusabira Orange 1:500 (MBL International, Cat# PM051), rat anti-BrdU 1:2500 (Accurate Chemical, Cat# OBT0030G), mouse anti-NeuN 1:1000 (Millipore, Cat# MAB377). Before incubation in blocking solution, tissue being stained with anti-BrdU antibody underwent antigen retrieval by pre-treatment with 2N hydrochloric acid (HCl) at 37 °C for 30 minutes, followed by 0.1M boric acid at room temperature for 10 minutes and three PBS washes. Tissue was incubated with primary antibodies at 4 °C overnight or for 40 hours with anti-BrdU. Following primary antibody incubation, tissue was triple washed in PBS and incubated for one hour with donkey fluorescent secondary antibodies (Jackson ImmunoResearch) diluted 1:400 in PBS with Hoechst 1:10,000 (ThermoFisher Scientific, Cat# 33342). After secondary antibody incubation, tissue was triple washed in PBS, mounted onto slides and coverslipped with Aqua-Poly/Mount (VWR, Cat# 87001-902).

All steps of the ISA were as described in [26 (link)], except that 5 μCi/ml [γ-³²P] ATP was used. In short, protein extracts were generated from TCA-treated cells. For every sample, protein concentration was determined by Coomassie blue to allow loading of equal amounts of proteins on 10% SDS-polyacrylamide gels along with 5μl of a standard containing a known amount of MMS activated Rad53 (“+ control”). After gel electrophoresis proteins were transferred to PVDF filters (Immobilon-P, Millipore membranes). Filters were subjected to a denaturation/renaturation protocol before the autophosphorylation reaction was performed by incubating membranes in kinase buffer in the presence of [γ-³²P] ATP. Dried filters were exposed on a Typhoon Trio+. After exposure, filters were re-probed with goat anti-Mcm2 (Santa Cruz) to check loading and allow comparison among different gels and mutants. Experiments were performed 2–3 times with similar results. MMS control (“+ control”): An Ay-120 culture (wt) with a density of 0.4 x 10⁷ cells/ml was treated with 0.1% MMS for ~60 minutes and harvested.