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. 2021 Jun 3;12:3318. doi: 10.1038/s41467-021-23580-4

Fig. 1. RNApII-pSer2 staining in dormant stem cells and characterization of the CDK9 substrate.

Fig. 1

a–c Immunofluorescence of frozen section from mouse brain subventricular zone (a), FACS-purified CD150+ CD34- KSL mouse hematopoietic stem cells (b), and frozen section from small intestine of Lgr5-eGFP mice (c). Antibodies were used against RNApII-pSer2, GFAP (glial fibrillary acidic protein) as a neural stem cell marker, GFP as a marker of LGR5-positive stem cells, and EpCAM, a marker of epithelial cells. In all cases, examples of RNApII-pSer2low cells are indicated by empty arrowheads. Filled arrowhead indicates a RNApII-pSer2high CD150+ CD34− stem cell in hematopoietic lineage (Fig. 1b) and Lgr5+ intestinal stem cells (Fig. 1c). d In vitro phosphorylation of bacterially expressed and purified GST-NPKATPPQI fusion protein by indicated cyclin-dependent kinases. Empty GST protein served as negative control for CDK9 kinase. e In vitro kinase assay using CDK9/CCNT1 and bacterially expressed and purified Venus into which NPKATPPQI was inserted at the indicated positions. WT (wild-type) Venus served as negative control. f Fluorescence of bacterially expressed and purified WT Venus and derivatives with the NPKATPPQI peptide inserted at indicated positions; concentration of all proteins was 2 mg/ml. g Comparison of relative fluorescence of WT Venus and VeN90 expressed in 501mel human melanoma cells. Two-tailed Student’s t-test ***p < 0.0001 (p value = 0.0001), error bars indicate SEM, n = 3 independent batch of cell line per group were analyzed. h Effect of 0.5 μM Flavopiridol (FVP) on fluorescence of Ve WT and VeN90 in 501mel cells. Cells were transfected with VeN90 expression vector and 24 h later treated with 0.5 μM FVP for 48 h. Two-tailed Student’s t-test ***p < 0.0001 (p value= 4.35061E-06), error bars indicate SEM, n = 3 independent batch of cell line per group were analyzed. i Western blot using indicated antibodies of extracts from 501mel cells treated with 0.5 μM FVP for 48 h. j Western blot of immunoprecipitated WT Venus or VeN90 expressed in Skmel-28 cells. Immunoprecipitates were subjected to treatment with 400 units of Lambda phosphatase (PPase) before being analyzed using Phos-Tag SDS-PAGE. k Western blot of immunoprecipitated VeN90 expressed in 501mel cells previously treated with 0.3 μM FVP for 48 h. Where indicated, immunoprecipitates were subjected to treatment with 400 units of Lambda phosphatase (PPase) before being analyzed using Phos-Tag SDS-PAGE. All the experiments were repeated at least three times with similar results. Source data are provided as a Source Data file.