Fig. 4

NEAT1–1 activated Pol II Ser2 phosphorylation through m6A in vitro and in prostate cancer. a Left, Coomassie blue staining of GST and GST-Pol II-C terminal domain (GST-Pol II-C, 372 amino acids of the Pol II-C terminal domain) recombinant proteins used for kinase assay. A star indicate the GST and GST-Pol II-C recombinant protein bands. Right, determination of CTD kinase (CDK19) activity. 1 μg GST or GST-Pol II-C terminal fusion proteins (in all lanes) and 2 μg NEAT1–1 RNA (lane 5) were incubated with CDK19 (lane 2,4,5) and CYCLINL1 produced from Quick coupled transcription/translation kit. After extensive washing, GST-beads were collected and subject to SDS/PAGE and western blot with Pol II Ser-2p antibodies. b Expression of Pol II Ser-2, Sre-5 phosphorylations and total Pol II proteins were measured by western blot in P-18 cells infected with control or NEAT1-specific siRNAs or in transfected with control or PSA eRNA-specific ASOs. c Expression of Pol II Ser-2 phosphorylations and total Pol II proteins were measured by western blot in P-18 cells with control siRNAs and siRNA pool of NEAT1, MALAT1, Xwast or ARLNC1. d Expression of Pol II Ser-2 phosphorylations and total Pol II proteins were measured by western blot in P-18 NEAT1–1-KO cells infected with control or NEAT1–1-overexpressed plasmids or NEAT1–1-m6A-mutants plasmids