anti ha  (Bethyl)

Journal: bioRxiv

Article Title: RNA polymerase III transcription-associated polyadenylation promotes the accumulation of noncoding retrotransposons during infection

doi: 10.1101/2025.05.14.654096

Figure Lengend Snippet: B2 SINE RNA polyadenylation during MHV68 infection requires mRNA cleavage and polyadenylation factors (A) NIH3T3 cells were mock-treated or infected with MHV68 at an MOI of 5. At 24 hpi, cells were harvested and lysed to extract total RNA. RNA was then digested with RNaseH in the presence or absence of oligo(dT) and subjected to Northern blotting using a primer specific to B2 SINE ncRNAs. SybrSafe stained gel shows 5.8S and 5S rRNA species in the bottom panel as loading controls. (B) NIH3T3 cells transfected with control non-targeting, or Nudt21 targeting siRNAs were mock-treated or infected with MHV68 at an MOI of 5. At 24hpi, cells were harvested and lysed to extract total protein and were analyzed by Western blotting with antibodies against Nud21and Vinculin (loading control). (C) Cells from panel B were also harvested for total RNA extraction and subjected to Northern blotting using a primer specific to B2 SINE ncRNAs. SybrSafe stained gel shows 5.8S and 5S rRNA species in the bottom panel as loading controls. (D) Total RNA extracted in panel C was also used for primer extension using primers specific to B2 SINE ncRNAs or 7SK RNA (control). The relative induction of B2 SINE ncRNAs for each sample was measured as the ratio of the mean integrated intensity between 7SK RNA and B2 SINE ncRNA levels and normalized to the mock-treated controls. (E) NIH3T3 cells transfected with control non-targeting, CPSF30 targeting, or CPSF73 targeting siRNAs were mock-treated or infected with MHV68 at an MOI of 5. At 24hpi, cells were harvested and lysed to extract total protein and were analyzed by Western blotting with antibodies against CPSF30, CPSF73, and Vinculin (loading control). (F) Cells from panel E were also harvested for total RNA extraction and subjected to Northern blotting using a primer specific to B2 SINE ncRNAs. SybrSafe stained gel shows 5.8S and 5S rRNA species in the bottom panel as loading controls. (G) Total RNA extracted in panel F was also used for primer extension using primers specific to B2 SINE ncRNAs or 7SK RNA (control). The relative induction of B2 SINE ncRNAs for each sample was measured as the ratio of the mean integrated intensity between 7SK RNA and B2 SINE ncRNA levels and normalized to the mock-treated controls.

Article Snippet: Blots were incubated in 5% milk in TBS with 0.1% Tween 20 (TBS-T) to block, followed by incubation with primary antibodies against anti-Nudt21 (Proteintech 10322-2-AP, 1;1000), anti-Vinculin antibody (abcam ab91459, 1;5,000), anti-CPSF30 antibody (Proteintech 15023-1-1AP, 1;1000), anti-CPSF73 antibody (Bethyl Laboratories A301-090A, 1;1000 or abcam ab72295, 1;100), anti-CPSF100 antibody, or anti-FIP1 antibody.

Techniques: Infection, Northern Blot, Staining, Transfection, Control, Western Blot, RNA Extraction

Journal: bioRxiv

Article Title: RNA polymerase III transcription-associated polyadenylation promotes the accumulation of noncoding retrotransposons during infection

doi: 10.1101/2025.05.14.654096

Figure Lengend Snippet: B2 SINE RNAs interact with mRNA 3 ′ -end processing factors during MHV68 infection (A) Schematic of comprehensive identification of RNA-binding proteins (ChIRP) protocol to detect B2 SINE ncRNA-protein interactions during MHV68 infection. (B) NIH3T3 cells were mock-treated or infected with MHV68 at an MOI of 5. At 24 hpi, ChIRP was performed using biotinylated B2 SINE oligos. Antisense LacZ oligos were used as a negative control. Protein was extracted from ChIRP samples and analyzed by Western blotting with antibodies against CPSF100, CPSF73, CPSF30, and FIP1. (C) Total protein samples from panel (B) were also subjected to mass-spectrometry. The table shows peptide counts and percent coverage detected in samples associated with mRNA CPSF machinery and other 3′ processing factors.

Article Snippet: Blots were incubated in 5% milk in TBS with 0.1% Tween 20 (TBS-T) to block, followed by incubation with primary antibodies against anti-Nudt21 (Proteintech 10322-2-AP, 1;1000), anti-Vinculin antibody (abcam ab91459, 1;5,000), anti-CPSF30 antibody (Proteintech 15023-1-1AP, 1;1000), anti-CPSF73 antibody (Bethyl Laboratories A301-090A, 1;1000 or abcam ab72295, 1;100), anti-CPSF100 antibody, or anti-FIP1 antibody.

Techniques: Infection, RNA Binding Assay, Negative Control, Western Blot, Mass Spectrometry

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

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: Two Tailed Test