Fig. 3: Nuclear class 3 PI3K interacts with RNA Pol2.
From: Class 3 PI3K coactivates the circadian clock to promote rhythmic de novo purine synthesis
a, Immunoblot analysis of soluble nuclear and cytosolic fractions from MEFs using antibodies to the indicated proteins. Tubulin and lamin A/C serve as controls for loading and fraction cross-contamination. The experiment was performed seven times. b, Immunofluorescence microscopy analyses of Vps15 in MEF cells treated with CSK buffer for 2 min. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 10 µm. c, Transcription assay with ectopically expressed VPS15–Flag and VPS34–Flag in MEFs. To label de novo transcription sites, MEFs were incubated with BrUTP 24 h post transfection. For co-localization, immunofluorescence microscopy was performed with anti-Flag and anti-BrUTP. Scale bar, 5 µm. d, Immunoblot analyses, using antibodies to the indicated proteins, of the Flag-containing immunoprecipitates from the soluble nuclear fraction of HEK293T cells transfected with empty vector (EV), or VPS15–Flag- or VPS34–Flag-expressing vectors. e, Immunofluorescence microscopy analyses of the co-localization of endogenous RNA Pol2 phospho-S5 (pS5), Vps15 and de novo transcription sites labelled with BrUTP in primary mouse hepatocytes. Scale bars, 10 µm and 5 µm (inset). c,e, The white triangles point to co-localization. f, Immunoblot analyses, using anti-RNA Pol2, of Vps15-containing immunoprecipitates from the soluble nuclear fractions of MEF cells. b–f, The experiments were performed three times. g, Immunoblot analyses of VPS15-containing complexes immunoprecipitated from HEK293T cells transiently transfected with Flag-conjugated WT or mutant VPS15 ((VPS15Mut1 (Mut1) and VPS15Mut2 (Mut2)). The cells were collected 48 h post transfection and VPS15 was immunoprecipitated using anti-Flag. The experiment was performed five times. Representative blots (a,d,f,g) or microscopy fields of view (b,c,e) are shown. IP, immunoprecipitation. Unprocessed blots are provided.
