anti cpsf73  (Bethyl)

Journal: PLOS ONE

Article Title: Isoginkgetin and Madrasin are poor splicing inhibitors

doi: 10.1371/journal.pone.0310519

Figure Lengend Snippet: (A) Schematic of the mNET-seq technique. (B) Metagene profile of total pol II mNET-seq treated with DMSO (blue) or 90 μM Madrasin (red) for 30 min on scaled expressed protein-coding genes. (C) qRT-PCR with primers amplifying different protein-coding genes. HeLa cells were treated with DMSO or 90 μM Madrasin for 30 min (red) or 60 min (orange). cDNA was generated with random hexamers. Values are normalised to the 7SK snRNA and shown as relative to DMSO, mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001. (D) Screenshot of the genome browser total pol II mNET-seq DMSO (blue) and Madrasin (red) tracks on the protein-coding gene KPNB1 . The arrow indicates the sense of transcription. (E) Total pol II, SPT5, CDC73, and CPSF73 ChIP-qPCR across the protein-coding gene KPNB1 in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001. (F) Ratios of SPT5 / total pol II, CDC73 / total pol II, or CPSF73 / total pol II from ChIP-qPCR on the intron-containing gene KPNB1 in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Article Snippet: For each IP, 60 μg of chromatin was incubated overnight on a rotating wheel at 4°C with the following antibodies: normal rabbit IgG (2729S, Cell Signaling), RNA polymerase II antibody (NBP2-32080, Novus Biologicals), Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (13499S, Cell Signaling Technology), Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (13523S, Cell Signaling Technology), CDC73 (A300-170A, Bethyl Laboratories), SUPT5H antibody (A300-868A, Bethyl Laboratories), and CPSF73 (A301-091A, Bethyl Laboratories).

Techniques: Quantitative RT-PCR, Generated, Two Tailed Test, ChIP-qPCR

Journal: PLOS ONE

Article Title: Isoginkgetin and Madrasin are poor splicing inhibitors

doi: 10.1371/journal.pone.0310519

Figure Lengend Snippet: (A) Metagene profiles of total pol II mNET-seq performed in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red) on scaled expressed intronless genes. (B) Screenshot of the genome browser total pol II mNET-seq DMSO (blue) and Madrasin (red) tracks of the intronless gene JUN . The arrow indicates the sense of transcription. (C) Total pol II, SPT5, CDC73, and CPSF73 ChIP-qPCR on the intronless gene JUN in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. (D) Metagene profiles of total pol II mNET-seq performed in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red) on scaled expressed histone genes. (E) Screenshot of the genome browser total pol II mNET-seq DMSO (blue) and Madrasin (red) tracks of the histone gene H1-2 . The arrow indicates the sense of transcription. (F) Total pol II, SPT5, CDC73, and CPSF73 ChIP-qPCR on the histone gene H1-2 in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05.

Article Snippet: For each IP, 60 μg of chromatin was incubated overnight on a rotating wheel at 4°C with the following antibodies: normal rabbit IgG (2729S, Cell Signaling), RNA polymerase II antibody (NBP2-32080, Novus Biologicals), Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (13499S, Cell Signaling Technology), Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (13523S, Cell Signaling Technology), CDC73 (A300-170A, Bethyl Laboratories), SUPT5H antibody (A300-868A, Bethyl Laboratories), and CPSF73 (A301-091A, Bethyl Laboratories).

Techniques: ChIP-qPCR, Two Tailed Test