![]() (d) NEAT1–1, MALAT1, Xist and ARLNC1 expressions were measured by qRT-PCR in P-18 primary cells. (c) NEAT1–1 RNA (green) and AR RNA (red) were stained using probes with FITC or TEXAS-RED dye. After extensive washing, GST-beads were collected and subject to SDS/PAGE and western blot with Flag, Myc, Pol II Ser-2p, total Pol II and GST antibodies. (b) GST or GST-Pol II-C terminal fusion proteins and 2 μg NEAT1–1 RNA were incubated with CDK19 and CYCLINL1 WT or mutated proteins produced from Quick coupled transcription/translation kit from T7 promoter. After extensive washing, GST-beads were collected and subject to SDS/PAGE and western blot with Pol II Ser-2p, total Pol II and GST antibodies. Right panel, 1 μg GST or GST-Pol II-C terminal fusion proteins and 2 μg NEAT1–1 RNA were incubated with CDK19 and CYCLINL1 produced from Quick coupled transcription/translation kit. ![]() Methylene blue was used to measure input loading. ![]() (a) Left panel, dot blot of m6A to testing NEAT1–1 full length RNA using anti-m6A. NEAT1–1 interacted with CYCLINL1 and CDK19. Analyswas of Tianjin Medical University data sets for levels of CYCLINL1 and CDK19 RNA were based on the RT-PCR. (e and f) Box and whisker plot showing CYCLINL1 and CDK19 signals upregulated in bone metastatic prostate cancer tissues. (d) CYCLINL1 and CDK19 RNA levels in normal human tissues. (c) Dot blot of m6A and biotin to testing NEAT1–1 WT and m6A-deletion probes using anti-m6A and anti-biotin antibodies. (b) Amino acid sequence comparwason of CDK8 and CDK19. (a) Expression of METT元 and Vinculin proteins were measured by western blot in P-18 METT元-KO cells infected with control or METT元 KO CRWASPR-Cas9 plasmids. Error bars represent mean ± SD for triplicate experiments. Means and standard deviations (error bar) were determined from three replicates. Transfected NEAT1–1 expressions were measured by qRT-PCR using primers targeting NEAT1–1 and plasmid in P-18 primary cells. (g) NEAT1–1 expressions were measured by qRT-PCR in 293 T and P-18 primary cells. m6A putative motif and sequences were shown in each figures. (c-f) Secondary structure of NEAT1–1 predicted by. M6A levels of NEAT1–1 and NEAT1–2 were measured by m6A-RIP-PCR in P-18 primary cells. (b) NEAT1–1 and NEAT1–2 expressions were measured by qRT-PCR in P-18 primary cells. The m6A profiles of FRMD8 were shown in genome browser. (a) m6A RIP-seq analyswas of m6A sites of FRMD8 by two independent antibodies. m6A sites and secondary structure in NEAT1. (c) Kaplan-Meier survival analyswas of the TCGA data set for the relationship between the levels of NEAT, expression of NEAT1–1 and survival time in prostate cancers. Analyswas of Tianjin Medical University data sets with fresh samples for levels of m6A and NEAT1 RNA were based on the m6A-RIP and RT-PCR. (b) Box and whisker plot showing NEAT1 m6A signals upregulated in bone metastatic prostate cancer tissues. (a) Box and whisker plot showing NEAT1–1 RNA signals upregulated in prostate cancer tissues compwered to normal tissues in TCGA data set. m6A of NEAT1–1 was elevated in prostate cancer and was a negative prognostic factor for patients. ![]() The Creative Commons Public Domain Dedication waiver ( ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.Īdditional file 1: Supplemental Fig. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. ![]() The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. ![]()
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