affinity purified bethyl laboratories a301 091a chip Search Results


cpsf2 antibody  (Bio-Techne corporation)
90
Bio-Techne corporation cpsf2 antibody
Cpsf2 Antibody, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit polyclonal anti cpsf3  (Bethyl)
94
Bethyl rabbit polyclonal anti cpsf3
Rabbit Polyclonal Anti Cpsf3, supplied by Bethyl, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cpsf1 3  (Proteintech)
93
Proteintech cpsf1 3
Cpsf1 3, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cpsf1 3  (Bethyl)
93
Bethyl cpsf1 3
Cpsf1 3, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cpsf73  (Bethyl)
93
Bethyl cpsf73
( A ) Average profile of RNAPII spanning the poly(A) site to the HMM extension termination of 1315 IRT genes in INTS11-depleted cells. ( B ) Average profile of pSer 2 -RNAPII spanning the poly(A) site to the HMM extension termination of 1315 IRT genes in INTS11-depleted cells. ( C ) Average profile of CSTF64 spanning the poly(A) site plus additional 10-kb downstream from N - TES. Profile at IRT genes without and with INTS11 shRNA induction. ( D ) Average profile of <t>CPSF73</t> spanning the poly(A) site plus additional 10-kb downstream from TES. Profile at IRT genes without and with INTS11 shRNA induction. ( E ) Boxplot of IRT gene groups separated by PRO-seq extension strength. ( F ) Boxplot of the extension length ratio in IRT gene groups separated by PRO-seq extension strength. ( G ) Boxplot of the RNAPII extension strength in IRT gene groups separated by PRO-seq extension strength. ( H ) Boxplot of the pSer 2 -RNAPII extension strength in IRT gene groups separated by PRO-seq extension strength.
Cpsf73, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/cpsf73/product/Bethyl
Average 93 stars, based on 1 article reviews
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fip1 a301-091a  (Bethyl)
90
Bethyl fip1 a301-091a
(A) HeLa nuclear extract (NE) or recombinant CFIm25-68 or CFIm25-59 complexes purified from baculovirus-infected Sf9 insect cells with or without alkaline phosphatase (CIP) treatment, were resolved by Phos-tag gel and analyzed by western blotting. The red arrows point to the phosphorylated proteins and the green arrows dephosphorylated proteins. (B) CFIm59 and CFIm68 RS domain sequences. (C) GST pulldown assay with GST, GST-RS(CFIm59) or (CFIm68) (purified from Sf9 cells) and in vitro translated 35S-labeled individual CPSF and CstF subunits. GST pulldown samples were resolved on SDS-PAGE and visualized by phosphorimaging (top panel). The same pulldown assay was performed with <t>6xHis-Fip1</t> expressed in Sf9 cells and pulldown samples were resolved on SDS-PAGE and analyzed by western blotting (lower panel). (D) A diagram of the Fip1 domain/regions. The Fip1-N and -C fragments were marked. Pulldown assays were similar to (C) with in vitro translated and 35S-labeled Fip1-N and Fip1-C. (E) Top panel: the sequences of the Fip1-RD and -RA peptides. Lower panel: Fip1-RD and –RA pulldown with purified 6xHis-CFIm25 (E. coli), 6xHis-CFIm25-59 (Sf9), and 6xHis-CFIm25-68 complexes (Sf9) and the bound proteins were resolved on SDS-PAGE and analyzed by western blotting. Negative control: streptavidin beads (beads). (F) Top panel: GST-RS(CFIm59/68) purified from E. coli were mock treated (−) or treated (+) with SRPK1 and then used in pulldown assays with 6xHis-Fip1. The pulldown samples were analyzed by western blotting. Lower panel: GST-RS(CFIm59/68) purified from Sf9 cells were mock untreated (−) or treated (+) with CIP, and then used in pulldown assays with purified 6xHis-Fip1. (G) Nuclear extracts from control, CFIm59-KO, or CFIm68-KO HEK293T cell lines were used for IP with anti-CFIm25 antibody and the IP samples were analyzed by western blotting. The red arrows mark the CFIm59 or CFIm68 that are absent in KO cell lines.
Fip1 A301 091a, supplied by Bethyl, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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c7orf50  (Bethyl)
90
Bethyl c7orf50
e, Ribo-seq and RNA-Seq read coverage, centered on the canonical start codon (ATG), for all genes in control and NF-YA KD ESCs. Triplet phasing, beginning at the canonical start codon, is observed for Ribo-Seq but not RNA-Seq data. RPKM, reads per kilobase per million mapped reads. f, Genome browser shot of NF-Y target gene <t>C7orf50</t> (3110082I17Rik) showing ribosome-protected RNA expression, as measured using Ribo-Seq, in control (blue) and NF-YA KD (red) ESCs. Also shown are tracks for RNA-Seq and Start-Seq in control and NF-YA KD ESCs. g, Western-blot analysis of NF-YA, Ppp2r5d, Tmx2, Atp5g1, Nono, C7orf50, Orc6, Shc1 and Khsrp in control and NF-YA knock-down (KD) ESCs 48h after siRNA transfection. Ran or Gapdh used as loading controls. Representative images are shown. h, Relative changes in gene expression (mRNA, orange) and protein levels (blue) in NF-YA KD vs control ESCs. Protein levels were determined using Western-blot analysis (see ), quantified by Licor Image Studio ® software. mRNA data normalized to Actin, HAZ and TBP . Protein data normalized to Ran or Gapdh. Error bars, SEM of three to five biological replicates. *P-value < 0.00002 (Student’s t-test, two-sided) i, Scatter plot showing the (RNA-Seq) expression of NF-Y bound (red) and non-NF-Y bound (black) genes in control (x-axis) and NF-YA KD (y-axis) ESCs.
C7orf50, supplied by Bethyl, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/c7orf50/product/Bethyl
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ints11 hpa029025 antibody  (Atlas Antibodies)
90
Atlas Antibodies ints11 hpa029025 antibody
e, Ribo-seq and RNA-Seq read coverage, centered on the canonical start codon (ATG), for all genes in control and NF-YA KD ESCs. Triplet phasing, beginning at the canonical start codon, is observed for Ribo-Seq but not RNA-Seq data. RPKM, reads per kilobase per million mapped reads. f, Genome browser shot of NF-Y target gene <t>C7orf50</t> (3110082I17Rik) showing ribosome-protected RNA expression, as measured using Ribo-Seq, in control (blue) and NF-YA KD (red) ESCs. Also shown are tracks for RNA-Seq and Start-Seq in control and NF-YA KD ESCs. g, Western-blot analysis of NF-YA, Ppp2r5d, Tmx2, Atp5g1, Nono, C7orf50, Orc6, Shc1 and Khsrp in control and NF-YA knock-down (KD) ESCs 48h after siRNA transfection. Ran or Gapdh used as loading controls. Representative images are shown. h, Relative changes in gene expression (mRNA, orange) and protein levels (blue) in NF-YA KD vs control ESCs. Protein levels were determined using Western-blot analysis (see ), quantified by Licor Image Studio ® software. mRNA data normalized to Actin, HAZ and TBP . Protein data normalized to Ran or Gapdh. Error bars, SEM of three to five biological replicates. *P-value < 0.00002 (Student’s t-test, two-sided) i, Scatter plot showing the (RNA-Seq) expression of NF-Y bound (red) and non-NF-Y bound (black) genes in control (x-axis) and NF-YA KD (y-axis) ESCs.
Ints11 Hpa029025 Antibody, supplied by Atlas Antibodies, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ints11 hpa029025 antibody - by Bioz Stars, 2026-03
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lamin a 39961 antibody  (Active Motif)
90
Active Motif lamin a 39961 antibody
e, Ribo-seq and RNA-Seq read coverage, centered on the canonical start codon (ATG), for all genes in control and NF-YA KD ESCs. Triplet phasing, beginning at the canonical start codon, is observed for Ribo-Seq but not RNA-Seq data. RPKM, reads per kilobase per million mapped reads. f, Genome browser shot of NF-Y target gene <t>C7orf50</t> (3110082I17Rik) showing ribosome-protected RNA expression, as measured using Ribo-Seq, in control (blue) and NF-YA KD (red) ESCs. Also shown are tracks for RNA-Seq and Start-Seq in control and NF-YA KD ESCs. g, Western-blot analysis of NF-YA, Ppp2r5d, Tmx2, Atp5g1, Nono, C7orf50, Orc6, Shc1 and Khsrp in control and NF-YA knock-down (KD) ESCs 48h after siRNA transfection. Ran or Gapdh used as loading controls. Representative images are shown. h, Relative changes in gene expression (mRNA, orange) and protein levels (blue) in NF-YA KD vs control ESCs. Protein levels were determined using Western-blot analysis (see ), quantified by Licor Image Studio ® software. mRNA data normalized to Actin, HAZ and TBP . Protein data normalized to Ran or Gapdh. Error bars, SEM of three to five biological replicates. *P-value < 0.00002 (Student’s t-test, two-sided) i, Scatter plot showing the (RNA-Seq) expression of NF-Y bound (red) and non-NF-Y bound (black) genes in control (x-axis) and NF-YA KD (y-axis) ESCs.
Lamin A 39961 Antibody, supplied by Active Motif, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
lamin a 39961 antibody - by Bioz Stars, 2026-03
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cpsf7 antibody  (Bethyl)
90
Bethyl cpsf7 antibody
e, Ribo-seq and RNA-Seq read coverage, centered on the canonical start codon (ATG), for all genes in control and NF-YA KD ESCs. Triplet phasing, beginning at the canonical start codon, is observed for Ribo-Seq but not RNA-Seq data. RPKM, reads per kilobase per million mapped reads. f, Genome browser shot of NF-Y target gene <t>C7orf50</t> (3110082I17Rik) showing ribosome-protected RNA expression, as measured using Ribo-Seq, in control (blue) and NF-YA KD (red) ESCs. Also shown are tracks for RNA-Seq and Start-Seq in control and NF-YA KD ESCs. g, Western-blot analysis of NF-YA, Ppp2r5d, Tmx2, Atp5g1, Nono, C7orf50, Orc6, Shc1 and Khsrp in control and NF-YA knock-down (KD) ESCs 48h after siRNA transfection. Ran or Gapdh used as loading controls. Representative images are shown. h, Relative changes in gene expression (mRNA, orange) and protein levels (blue) in NF-YA KD vs control ESCs. Protein levels were determined using Western-blot analysis (see ), quantified by Licor Image Studio ® software. mRNA data normalized to Actin, HAZ and TBP . Protein data normalized to Ran or Gapdh. Error bars, SEM of three to five biological replicates. *P-value < 0.00002 (Student’s t-test, two-sided) i, Scatter plot showing the (RNA-Seq) expression of NF-Y bound (red) and non-NF-Y bound (black) genes in control (x-axis) and NF-YA KD (y-axis) ESCs.
Cpsf7 Antibody, supplied by Bethyl, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/cpsf7 antibody/product/Bethyl
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cpsf7 antibody - by Bioz Stars, 2026-03
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wdr33  (Bethyl)
93
Bethyl wdr33
e, Ribo-seq and RNA-Seq read coverage, centered on the canonical start codon (ATG), for all genes in control and NF-YA KD ESCs. Triplet phasing, beginning at the canonical start codon, is observed for Ribo-Seq but not RNA-Seq data. RPKM, reads per kilobase per million mapped reads. f, Genome browser shot of NF-Y target gene <t>C7orf50</t> (3110082I17Rik) showing ribosome-protected RNA expression, as measured using Ribo-Seq, in control (blue) and NF-YA KD (red) ESCs. Also shown are tracks for RNA-Seq and Start-Seq in control and NF-YA KD ESCs. g, Western-blot analysis of NF-YA, Ppp2r5d, Tmx2, Atp5g1, Nono, C7orf50, Orc6, Shc1 and Khsrp in control and NF-YA knock-down (KD) ESCs 48h after siRNA transfection. Ran or Gapdh used as loading controls. Representative images are shown. h, Relative changes in gene expression (mRNA, orange) and protein levels (blue) in NF-YA KD vs control ESCs. Protein levels were determined using Western-blot analysis (see ), quantified by Licor Image Studio ® software. mRNA data normalized to Actin, HAZ and TBP . Protein data normalized to Ran or Gapdh. Error bars, SEM of three to five biological replicates. *P-value < 0.00002 (Student’s t-test, two-sided) i, Scatter plot showing the (RNA-Seq) expression of NF-Y bound (red) and non-NF-Y bound (black) genes in control (x-axis) and NF-YA KD (y-axis) ESCs.
Wdr33, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/wdr33/product/Bethyl
Average 93 stars, based on 1 article reviews
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Image Search Results


( A ) Average profile of RNAPII spanning the poly(A) site to the HMM extension termination of 1315 IRT genes in INTS11-depleted cells. ( B ) Average profile of pSer 2 -RNAPII spanning the poly(A) site to the HMM extension termination of 1315 IRT genes in INTS11-depleted cells. ( C ) Average profile of CSTF64 spanning the poly(A) site plus additional 10-kb downstream from N - TES. Profile at IRT genes without and with INTS11 shRNA induction. ( D ) Average profile of CPSF73 spanning the poly(A) site plus additional 10-kb downstream from TES. Profile at IRT genes without and with INTS11 shRNA induction. ( E ) Boxplot of IRT gene groups separated by PRO-seq extension strength. ( F ) Boxplot of the extension length ratio in IRT gene groups separated by PRO-seq extension strength. ( G ) Boxplot of the RNAPII extension strength in IRT gene groups separated by PRO-seq extension strength. ( H ) Boxplot of the pSer 2 -RNAPII extension strength in IRT gene groups separated by PRO-seq extension strength.

Journal: Science Advances

Article Title: Integrator enforces the fidelity of transcriptional termination at protein-coding genes

doi: 10.1126/sciadv.abe3393

Figure Lengend Snippet: ( A ) Average profile of RNAPII spanning the poly(A) site to the HMM extension termination of 1315 IRT genes in INTS11-depleted cells. ( B ) Average profile of pSer 2 -RNAPII spanning the poly(A) site to the HMM extension termination of 1315 IRT genes in INTS11-depleted cells. ( C ) Average profile of CSTF64 spanning the poly(A) site plus additional 10-kb downstream from N - TES. Profile at IRT genes without and with INTS11 shRNA induction. ( D ) Average profile of CPSF73 spanning the poly(A) site plus additional 10-kb downstream from TES. Profile at IRT genes without and with INTS11 shRNA induction. ( E ) Boxplot of IRT gene groups separated by PRO-seq extension strength. ( F ) Boxplot of the extension length ratio in IRT gene groups separated by PRO-seq extension strength. ( G ) Boxplot of the RNAPII extension strength in IRT gene groups separated by PRO-seq extension strength. ( H ) Boxplot of the pSer 2 -RNAPII extension strength in IRT gene groups separated by PRO-seq extension strength.

Article Snippet: The antibodies used in this study are as follows: for ChIP-seq, RPB1 NTD (Cell Signaling Technology, 14958, lot #1), RPB1 Ser 2 (Abcam, 5095, lot #GR3225147-1), H3K36me3 (Abcam, 9050, lot #GR3257952-1), H3K36me2 (Abcam, 9049, lot #GR3236147-1), CSTF64 (Bethyl Laboratories, A301-92A, lot #A301-092A-2), and CPSF73 (Bethyl Laboratories, A301-019A, lot #A301-091A-1); for Western blot, INTS11 (Atlas Antibodies, HPA029025, lot #A107128), lamin A (Active Motif, 39961, lot #33310001), TFIIB (Cell Signaling Technology, 4169s, lot #1), CPSF100 (Bethyl Laboratories, A301-583A-M, lot #A301-583A-M-1), CSTF50 (Bethyl Laboratories, A301-250A-M, lot #A301-250A-M-3), CSTF64 (Bethyl Laboratories, A301-092A-M, lot #A301-092A-M-2), CPSF73 (Bethyl Laboratories, A301-091A-M, lot #A301-091A-M-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, ab8245, lot #GR3317834-1).

Techniques: shRNA

( A ) 3′ RNA-seq Genome Browser example of the IRT gene DELE1 in WT INTS11 and E203Q INTS11 rescue cells. ( B ) CM of canonical versus downstream termination site usage in WT INTS11 rescue cells. ( C ) CM of canonical versus downstream termination site usage in E203Q INTS11 rescue cells. ( D ) Motif analysis at canonical 3′ end and downstream extended transcript 3′ end. A window of −/+150 nt from the peak was used for the analysis; top 5 motifs were shown. ( E ) CPSF73 ChIP-seq profiles at 3′ end in the downstream region of IRT genes. Profiles without (blue line) and with INTS11 shRNA induction (red line). ( F ) CSTF64 ChIP-seq profiles at 3′ end in the downstream region of IRT genes. Profiles without (blue line) and with INTS11 shRNA induction (red line).

Journal: Science Advances

Article Title: Integrator enforces the fidelity of transcriptional termination at protein-coding genes

doi: 10.1126/sciadv.abe3393

Figure Lengend Snippet: ( A ) 3′ RNA-seq Genome Browser example of the IRT gene DELE1 in WT INTS11 and E203Q INTS11 rescue cells. ( B ) CM of canonical versus downstream termination site usage in WT INTS11 rescue cells. ( C ) CM of canonical versus downstream termination site usage in E203Q INTS11 rescue cells. ( D ) Motif analysis at canonical 3′ end and downstream extended transcript 3′ end. A window of −/+150 nt from the peak was used for the analysis; top 5 motifs were shown. ( E ) CPSF73 ChIP-seq profiles at 3′ end in the downstream region of IRT genes. Profiles without (blue line) and with INTS11 shRNA induction (red line). ( F ) CSTF64 ChIP-seq profiles at 3′ end in the downstream region of IRT genes. Profiles without (blue line) and with INTS11 shRNA induction (red line).

Article Snippet: The antibodies used in this study are as follows: for ChIP-seq, RPB1 NTD (Cell Signaling Technology, 14958, lot #1), RPB1 Ser 2 (Abcam, 5095, lot #GR3225147-1), H3K36me3 (Abcam, 9050, lot #GR3257952-1), H3K36me2 (Abcam, 9049, lot #GR3236147-1), CSTF64 (Bethyl Laboratories, A301-92A, lot #A301-092A-2), and CPSF73 (Bethyl Laboratories, A301-019A, lot #A301-091A-1); for Western blot, INTS11 (Atlas Antibodies, HPA029025, lot #A107128), lamin A (Active Motif, 39961, lot #33310001), TFIIB (Cell Signaling Technology, 4169s, lot #1), CPSF100 (Bethyl Laboratories, A301-583A-M, lot #A301-583A-M-1), CSTF50 (Bethyl Laboratories, A301-250A-M, lot #A301-250A-M-3), CSTF64 (Bethyl Laboratories, A301-092A-M, lot #A301-092A-M-2), CPSF73 (Bethyl Laboratories, A301-091A-M, lot #A301-091A-M-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, ab8245, lot #GR3317834-1).

Techniques: RNA Sequencing, ChIP-sequencing, shRNA

(A) HeLa nuclear extract (NE) or recombinant CFIm25-68 or CFIm25-59 complexes purified from baculovirus-infected Sf9 insect cells with or without alkaline phosphatase (CIP) treatment, were resolved by Phos-tag gel and analyzed by western blotting. The red arrows point to the phosphorylated proteins and the green arrows dephosphorylated proteins. (B) CFIm59 and CFIm68 RS domain sequences. (C) GST pulldown assay with GST, GST-RS(CFIm59) or (CFIm68) (purified from Sf9 cells) and in vitro translated 35S-labeled individual CPSF and CstF subunits. GST pulldown samples were resolved on SDS-PAGE and visualized by phosphorimaging (top panel). The same pulldown assay was performed with 6xHis-Fip1 expressed in Sf9 cells and pulldown samples were resolved on SDS-PAGE and analyzed by western blotting (lower panel). (D) A diagram of the Fip1 domain/regions. The Fip1-N and -C fragments were marked. Pulldown assays were similar to (C) with in vitro translated and 35S-labeled Fip1-N and Fip1-C. (E) Top panel: the sequences of the Fip1-RD and -RA peptides. Lower panel: Fip1-RD and –RA pulldown with purified 6xHis-CFIm25 (E. coli), 6xHis-CFIm25-59 (Sf9), and 6xHis-CFIm25-68 complexes (Sf9) and the bound proteins were resolved on SDS-PAGE and analyzed by western blotting. Negative control: streptavidin beads (beads). (F) Top panel: GST-RS(CFIm59/68) purified from E. coli were mock treated (−) or treated (+) with SRPK1 and then used in pulldown assays with 6xHis-Fip1. The pulldown samples were analyzed by western blotting. Lower panel: GST-RS(CFIm59/68) purified from Sf9 cells were mock untreated (−) or treated (+) with CIP, and then used in pulldown assays with purified 6xHis-Fip1. (G) Nuclear extracts from control, CFIm59-KO, or CFIm68-KO HEK293T cell lines were used for IP with anti-CFIm25 antibody and the IP samples were analyzed by western blotting. The red arrows mark the CFIm59 or CFIm68 that are absent in KO cell lines.

Journal: Molecular cell

Article Title: Molecular mechanisms for CFIm-mediated regulation of mRNA alternative polyadenylation

doi: 10.1016/j.molcel.2017.11.031

Figure Lengend Snippet: (A) HeLa nuclear extract (NE) or recombinant CFIm25-68 or CFIm25-59 complexes purified from baculovirus-infected Sf9 insect cells with or without alkaline phosphatase (CIP) treatment, were resolved by Phos-tag gel and analyzed by western blotting. The red arrows point to the phosphorylated proteins and the green arrows dephosphorylated proteins. (B) CFIm59 and CFIm68 RS domain sequences. (C) GST pulldown assay with GST, GST-RS(CFIm59) or (CFIm68) (purified from Sf9 cells) and in vitro translated 35S-labeled individual CPSF and CstF subunits. GST pulldown samples were resolved on SDS-PAGE and visualized by phosphorimaging (top panel). The same pulldown assay was performed with 6xHis-Fip1 expressed in Sf9 cells and pulldown samples were resolved on SDS-PAGE and analyzed by western blotting (lower panel). (D) A diagram of the Fip1 domain/regions. The Fip1-N and -C fragments were marked. Pulldown assays were similar to (C) with in vitro translated and 35S-labeled Fip1-N and Fip1-C. (E) Top panel: the sequences of the Fip1-RD and -RA peptides. Lower panel: Fip1-RD and –RA pulldown with purified 6xHis-CFIm25 (E. coli), 6xHis-CFIm25-59 (Sf9), and 6xHis-CFIm25-68 complexes (Sf9) and the bound proteins were resolved on SDS-PAGE and analyzed by western blotting. Negative control: streptavidin beads (beads). (F) Top panel: GST-RS(CFIm59/68) purified from E. coli were mock treated (−) or treated (+) with SRPK1 and then used in pulldown assays with 6xHis-Fip1. The pulldown samples were analyzed by western blotting. Lower panel: GST-RS(CFIm59/68) purified from Sf9 cells were mock untreated (−) or treated (+) with CIP, and then used in pulldown assays with purified 6xHis-Fip1. (G) Nuclear extracts from control, CFIm59-KO, or CFIm68-KO HEK293T cell lines were used for IP with anti-CFIm25 antibody and the IP samples were analyzed by western blotting. The red arrows mark the CFIm59 or CFIm68 that are absent in KO cell lines.

Article Snippet: Fip1 , Bethyl , A301-091A; RRID:AB_2084528.

Techniques: Recombinant, Purification, Infection, Western Blot, GST Pulldown Assay, In Vitro, Labeling, SDS Page, Negative Control, Control

The solid red line with arrow indicates that CFIm helps to recruits CPSF through direct interactions and the dotted red line with arrow indicates that CFIm promotes CstF recruitment indirectly (A–B). The dotted grey lines indicate the lack of RE/D regions in the yeast Fip1 and Snp1 (C–D). UE: U-rich elements. CFIm25-68 is a dimer, but shown as a monomer due to space limitation (A). The blue arrows represent cleavage and the widths of the arrows represent the frequencies of PAS usage.

Journal: Molecular cell

Article Title: Molecular mechanisms for CFIm-mediated regulation of mRNA alternative polyadenylation

doi: 10.1016/j.molcel.2017.11.031

Figure Lengend Snippet: The solid red line with arrow indicates that CFIm helps to recruits CPSF through direct interactions and the dotted red line with arrow indicates that CFIm promotes CstF recruitment indirectly (A–B). The dotted grey lines indicate the lack of RE/D regions in the yeast Fip1 and Snp1 (C–D). UE: U-rich elements. CFIm25-68 is a dimer, but shown as a monomer due to space limitation (A). The blue arrows represent cleavage and the widths of the arrows represent the frequencies of PAS usage.

Article Snippet: Fip1 , Bethyl , A301-091A; RRID:AB_2084528.

Techniques:

KEY RESOURCES TABLE

Journal: Molecular cell

Article Title: Molecular mechanisms for CFIm-mediated regulation of mRNA alternative polyadenylation

doi: 10.1016/j.molcel.2017.11.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Fip1 , Bethyl , A301-091A; RRID:AB_2084528.

Techniques: Recombinant, Reporter Assay, Transfection, Cloning, In Vitro, Software

e, Ribo-seq and RNA-Seq read coverage, centered on the canonical start codon (ATG), for all genes in control and NF-YA KD ESCs. Triplet phasing, beginning at the canonical start codon, is observed for Ribo-Seq but not RNA-Seq data. RPKM, reads per kilobase per million mapped reads. f, Genome browser shot of NF-Y target gene C7orf50 (3110082I17Rik) showing ribosome-protected RNA expression, as measured using Ribo-Seq, in control (blue) and NF-YA KD (red) ESCs. Also shown are tracks for RNA-Seq and Start-Seq in control and NF-YA KD ESCs. g, Western-blot analysis of NF-YA, Ppp2r5d, Tmx2, Atp5g1, Nono, C7orf50, Orc6, Shc1 and Khsrp in control and NF-YA knock-down (KD) ESCs 48h after siRNA transfection. Ran or Gapdh used as loading controls. Representative images are shown. h, Relative changes in gene expression (mRNA, orange) and protein levels (blue) in NF-YA KD vs control ESCs. Protein levels were determined using Western-blot analysis (see ), quantified by Licor Image Studio ® software. mRNA data normalized to Actin, HAZ and TBP . Protein data normalized to Ran or Gapdh. Error bars, SEM of three to five biological replicates. *P-value < 0.00002 (Student’s t-test, two-sided) i, Scatter plot showing the (RNA-Seq) expression of NF-Y bound (red) and non-NF-Y bound (black) genes in control (x-axis) and NF-YA KD (y-axis) ESCs.

Journal: bioRxiv

Article Title: NF-Y controls fidelity of transcription initiation at gene promoters through maintenance of the nucleosome-depleted region

doi: 10.1101/369389

Figure Lengend Snippet: e, Ribo-seq and RNA-Seq read coverage, centered on the canonical start codon (ATG), for all genes in control and NF-YA KD ESCs. Triplet phasing, beginning at the canonical start codon, is observed for Ribo-Seq but not RNA-Seq data. RPKM, reads per kilobase per million mapped reads. f, Genome browser shot of NF-Y target gene C7orf50 (3110082I17Rik) showing ribosome-protected RNA expression, as measured using Ribo-Seq, in control (blue) and NF-YA KD (red) ESCs. Also shown are tracks for RNA-Seq and Start-Seq in control and NF-YA KD ESCs. g, Western-blot analysis of NF-YA, Ppp2r5d, Tmx2, Atp5g1, Nono, C7orf50, Orc6, Shc1 and Khsrp in control and NF-YA knock-down (KD) ESCs 48h after siRNA transfection. Ran or Gapdh used as loading controls. Representative images are shown. h, Relative changes in gene expression (mRNA, orange) and protein levels (blue) in NF-YA KD vs control ESCs. Protein levels were determined using Western-blot analysis (see ), quantified by Licor Image Studio ® software. mRNA data normalized to Actin, HAZ and TBP . Protein data normalized to Ran or Gapdh. Error bars, SEM of three to five biological replicates. *P-value < 0.00002 (Student’s t-test, two-sided) i, Scatter plot showing the (RNA-Seq) expression of NF-Y bound (red) and non-NF-Y bound (black) genes in control (x-axis) and NF-YA KD (y-axis) ESCs.

Article Snippet: The following antibodies were used in : Ppp2r5d (Bethyl, A301-100A), Tmx2 (Abcam, ab105675), Atp5g1 (Abcam, ab180149), Nono (Santa Cruz, sc-166702), C7orf50 (Bethyl, A305-091A), Orc6 (Santa Cruz, sc-390490), Shc1 (Bethyl, A302-019A), Khsrp (Bethyl, A302-021A) and Gapdh (Santa Cruz, sc-25778).

Techniques: RNA Sequencing Assay, RNA Expression, Western Blot, Transfection, Expressing, Software