Ant was used as Tip110-depleted Hela nuclear extract. Tip110 protein on the immunoabsorbed beads was eluted by adding 20 l of 4?SDS sample buffer, followed by boiling 5 min. The eluent and aliquot of the Tip110-depleted Hela nuclear extract were subjected to 10 SDSPAGE gel and Western blot analysis to ensure efficient Tip110 depletion.PremRNA splicingdependent CAT reporter gene assay293T cells were plated in a 6-well plate at a density of 0.72 ? 106 cells/well and 1 day later transfected with 1 g pDM138, 1 g pcRev, 1? g pTip110.His or pTip110.siRNA. In all transfections, 1-phenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole a CMVGal plasmid was included to normalize transfection variations, and pcDNA3 was used to equalize the amount of transfected DNA. Forty-eight hours after transfection, the cells were harvested and subjected to 3 rounds of freeze haw in 0.25 M Tris.HCl, pH 8.0, and the lysates were collected for -galactosidase and CAT activities. -galactosidase was determined using a colorimtric assay [20], while CAT was determined by one-step simple phase extraction and scintillation counting as described [33].Liu et al. Cell Biosci (2015) 5:Page 4 ofPreparation of cell lysates and Western blot analysisCells were washed twice with ice-cold phosphate-buffered saline (PBS), and cell pellets were suspended in two volumes of whole cell lysis buffer containing10 mM NaHPO4, 150 mM NaCl, 1 Triton X-100, 0.1 SDS, 0.2 sodium azide, 0.5 sodium deoxycholate, 0.004 sodium fluoride, and 1 mM sodium PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8711135 orthovanadate and incubated on ice for 10 min. Cell lysates were obtained by centrifugation, removal of the cell debris, and electrophoretically separated on 10 SDS-PAGE and analyzed by immunoblotting. Anti-His antibody was from Qiagen (Valencia, CA, USA), while -actin was from Santa Cruz Biotech. (Santa Cruz, CA, USA). The blots were first probed with primary antibodies, followed by appropriate horseradish peroxidase-conjugated secondary antibodies, and then visualized with the ECL system (Amersham).RNA isolation and RNASeqHela were transfected with human Tip110 siRNA or the universal control siRNA. Total RNA was purified from transfected cells using Trizol according to the manufacturer's instructions (Life Technologies, Grand Island, NY, USA) and used for oncotarget.13387 RNA-Seq. The RNA-Seq was performed at Tert-butyl 2-(chloromethyl)pyrrolidine-1-carboxylate the Genomic Core of UT Southwestern Medical Center, Dallas, TX, USA. The RNA-Seq reads were aligned to the human genome sequences for further differential and spliceosome pathway analysis using the Panther Pathway Analysis software.qRTPCRFigure 1 Identification of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13485127 U6 as a Tip110 binding RNA target. a Scheme of the yeast three-hybrid cloning strategy. LexA BD-MS2: LexA DNA binding domain fused with phage coat protein MS2; MS2 RNA NA library: the target RNA sequence of MS2 protein was fused to a pool of RNAs transcribed from human genomic DNA; Gal4 ADTip110: Gal4 activation domain fused to Tip110 protein. Respective binding of MS2 protein to MS2 target RNA and Tip110 protein to its putative RNAs leads to activation of LacZ and His reporter genes. b Sequences of RNA hits obtained from the yeast three-hybrid screening using Tip110 as the bait. The consensus sequence was matched to small nuclear U6 RNA at nucleotides 34?6.Total RNA was extracted using Trizol and used to synthesize cDNA using a ScriptII RT reagent kit (Promega). cDNAs were used for qPCR using a Power Sybr reen PCR kit (Life Technologies) according to the manufacturer's instructions. The qPCR program consisted of one cycle of 95 for 10.