Journal: The Journal of Cell Biology

Article Title: CDK2 regulates the NRF1/ Ehmt1 axis during meiotic prophase I

doi: 10.1083/jcb.201903125

Figure Lengend Snippet: NRF1 binds to CDK2 and is a novel CDK2 substrate in vitro. (A) HEK-293T cells expressing either empty vector control, NRF1-MYC, or CDK2-HA, or coexpressing NRF1-MYC and CDK2-HA were lysed as described. NRF1-MYC could be coimmunoprecipitated with CDK2-HA and vice versa (lane 4). Bottom panels show Western blots of 10% of total protein lysate used for immunoprecipitation (Input). HSP90 was used as a loading control. (B) Western blotting of immunoprecipitates from whole testis lysate. Lane 1: 1% of input (16 µg of whole testis lysate); lane 2: IP CDK2; lane 3: IP NRF1; lane 4: IP nonspecific IgG. Coimmunoprecipitation can be seen in lanes 2 and 3 of both of the upper and lower panels. (C) Evolutionary conservation of the amino acid sequence surrounding serines 102 and 136 of NRF1 in multiple species. Potential CDK phosphorylation sites are highlighted in pink. (D) Representative kinase assay using 1 µg of wild-type (lane 1), S136A mutant (lane 2), or S102A/S136A (lane 3) full-length NRF1–GST fusion proteins as a substrate of CDK2/cyclin A in the presence of radiolabeled ATP (upper panel). Quantification of the corresponding phospho-signal was done by PhosphoImager using the Multi Gauge software (Ver3.X) from three replicate experiments as displayed below. Significance was determined by one-way ANOVA. Data were assumed to be normally distributed, but this was not formally tested (****, P < 0.0001). Error bars are representative of the SD of phospho-signal values from at least three technical replicates of the same experiment. Equal protein loading is shown by Coomassie staining of the dried gel (lower panel). (E) EMSA using a probe of the Ehmt1 promoter. A DIG-labeled Ehmt1 promoter probe was incubated with increasing concentrations of either CDK2/cyclin A2 phosphorylated NRF1 (pNRF1; lanes 6–8; 1, 2, or 3 µg, respectively) or nonphosphorylated NRF1 (lanes 3–5; 1, 2, or 3 µg, respectively). “Probe only” and “Probe + Poly DIDC only” conditions are shown in lanes 1 and 2, respectively. Shifted probe (complexes) or free probe (probe) were detected via anti-DIG-alkaline phosphatase (AP) antibody treatment followed by chemiluminescent detection of alkaline phosphatase. Protein loading is shown by both Western blotting and Coomassie staining of NRF1 and pNRF1 on separate SDS-PAGE gels. All experiments were repeated at least three times with similar results. One representative image is shown in each figure. AU, arbitrary units.

Article Snippet: Kinase assays were quenched via the addition of 6× Laemmli SDS sample buffer to a final concentration of 1×, followed by boiling at 95°C for 5 min. Proteins were separated by SDS-PAGE on 8% polyacrylamide gels, and gels were dried using a Speedgel gel dryer (SG210D; Thermo Fisher Scientific) at 80°C for 2 h. Levels of incorporated radioactivity within dried gels were then quantified using a nonconfocal variable mode laser scanner/PhosphoImager (Typhoon FLA-7000; Fujifilm) and the Multi Gauge software (Ver3.X).

Techniques: In Vitro, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation, Sequencing, Kinase Assay, Mutagenesis, Software, Staining, Labeling, Incubation, SDS Page