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U2af1

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U2AF1 is a protein involved in the regulation of pre-mRNA splicing. It plays a critical role in the recognition and binding of the 3' splice site during the early stages of spliceosome assembly. U2AF1 is essential for the recruitment of the U2 snRNP to the pre-mRNA, a key step in the splicing process.

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Lab products found in correlation

Sf3b1, by Fortis Life Sciences (3 mentions) Vinculin, by Merck Group (2 mentions) Snrpf, by Abcam (2 mentions) Bud31, by Proteintech (2 mentions) Prp19, by Fortis Life Sciences (2 mentions) Sf3a1, by Fortis Life Sciences (2 mentions) Eftud2, by Fortis Life Sciences (2 mentions) Eif2s1, by Abcepta (2 mentions) Eif3i p36, by BioLegend (2 mentions) C myc, by Cell Signaling Technology (2 mentions) Flag tag (flag), by Merck Group (2 mentions) Cleaved caspase 3, by Cell Signaling Technology (2 mentions) Fluorescently conjugated secondary antibodies, by LI COR (1 mentions) Nupage sample buffer, by Thermo Fisher Scientific (1 mentions) Odyssey clx fluorescent scanner, by LI COR (1 mentions) Eif2α, by Cell Signaling Technology (1 mentions) Phospho eif2α ser51, by Cell Signaling Technology (1 mentions) Phospho 4e bp1 thr37 46, by Cell Signaling Technology (1 mentions) 4e bp1, by Cell Signaling Technology (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions)

4 protocols using u2af1

1

Western Blot Analysis of RNA-Binding Proteins

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Western blot analysis was performed using samples of whole cell lysate or eluate from immunoprecipitation prepared in NuPage sample buffer supplemented with antioxidant reagent according to manufacturer’s specifications (Invitrogen). Samples were heated (10’ @ 70°C), then separated by electrophoresis in NuPage 4–20% polyacrylamide gels and transferred to nitrocellulose membanes using the NuPage XCell II Blow Module (Invitrogen). Blots were visualized using the Li-COR Odyssey CLX fluorescent scanner after blocking with fluorescent blocking buffer (Li-COR Biosciences), incubation with primary antibodies and fluorescently conjugated secondary antibodies (Li-COR Biosciences).
Primary antibodies: Mouse monoclonal–HNRNPC1/C2 (sc-32308, Santa Cruz), HNRNPU (3C6, Millipore), SRSF3 (7B4, Millipore), SRSF1 (AK96, Millipore); Rabbit polyclonal–FUSIP1 (A302-282A-1,Bethyl), ILF2 (A303-147A-1, Bethyl), IgG (2729, Cell Signaling), U2AF1 (A302-079A-1, Bethyl), SAM68/KHDRBS1 (sc-333, Santa Cruz), SFRS10 (AV40528, Sigma).
Whisenant T.C., Peralta E.R., Aarreberg L.D., Gao N.J., Head S.R., Ordoukhanian P., Williamson J.R, & Salomon D.R. (2015). The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells. PLoS ONE, 10(12), e0144409.
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2

Comprehensive Protein Analysis by Western Blot

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Cells were lysed in 1× SDS sample buffer (62.5mM Tris-HCl, pH6.8, 10% glycerol, 2% SDS, 2.5% β-mercaptoethanol) and heated at 95°C for 12 minutes. The following antibodies were used for Western blotting: Flag (Sigma, A8592), BUD31 (ProteinTech, 11798-1-AP), SF3B1 (Bethyl, A300-996A), Prp19 (Bethyl, A300-101A), U2AF1 (Bethyl, A302-079A), SF3A1 (Bethyl, A301-603A), EFTUD2 (Bethyl, A300-957A), SNRPF (Abcam, 154870), HER2 (Millipore, 06-562), EGFR (Cell Signaling, 2232), cleaved caspase-3 (Cell Signaling, 9664), RPS8 (Assay Biotechnology, R12-3466), EIF2S1 (Abgent, AP13469s), eIF3I (p36) (Biolegend, 646701), and c-Myc (D84C12) (Cell Signaling, 5605). Vinculin (Sigma, V9131) and ran (BD Biosciences, 610340) was used as loading control.
Hsu T.Y., Simon L.M., Neill N., Marcotte R., Sayad A., Bland C.S., Echeverria G.V., Sun T., Kurley S.J., Tyagi S., Karlin K.L., Dominguez-Vidaña R., Hartman J.D., Renwick A., Scorsone K., Bernardi R.J., Skinner S.O., Jain A., Orellana M., Lagisetti C., Golding I., Jung S.Y., Neilson J.R., Zhang X.H., Cooper T.A., Webb T.R., Neel B.G., Shaw C.A, & Westbrook T.F. (2015). The spliceosome is a therapeutic vulnerability in MYC-driven cancer. Nature, 525(7569), 384-388.
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3

Comprehensive Protein Analysis by Western Blot

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Cells were lysed in 1× SDS sample buffer (62.5mM Tris-HCl, pH6.8, 10% glycerol, 2% SDS, 2.5% β-mercaptoethanol) and heated at 95°C for 12 minutes. The following antibodies were used for Western blotting: Flag (Sigma, A8592), BUD31 (ProteinTech, 11798-1-AP), SF3B1 (Bethyl, A300-996A), Prp19 (Bethyl, A300-101A), U2AF1 (Bethyl, A302-079A), SF3A1 (Bethyl, A301-603A), EFTUD2 (Bethyl, A300-957A), SNRPF (Abcam, 154870), HER2 (Millipore, 06-562), EGFR (Cell Signaling, 2232), cleaved caspase-3 (Cell Signaling, 9664), RPS8 (Assay Biotechnology, R12-3466), EIF2S1 (Abgent, AP13469s), eIF3I (p36) (Biolegend, 646701), and c-Myc (D84C12) (Cell Signaling, 5605). Vinculin (Sigma, V9131) and ran (BD Biosciences, 610340) was used as loading control.
Hsu T.Y., Simon L.M., Neill N., Marcotte R., Sayad A., Bland C.S., Echeverria G.V., Sun T., Kurley S.J., Tyagi S., Karlin K.L., Dominguez-Vidaña R., Hartman J.D., Renwick A., Scorsone K., Bernardi R.J., Skinner S.O., Jain A., Orellana M., Lagisetti C., Golding I., Jung S.Y., Neilson J.R., Zhang X.H., Cooper T.A., Webb T.R., Neel B.G., Shaw C.A, & Westbrook T.F. (2015). The spliceosome is a therapeutic vulnerability in MYC-driven cancer. Nature, 525(7569), 384-388.
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4

Molecular Characterization of Cellular Machinery

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The following antibodies and plasmids were used where indicated. SF3B1 (Bethyl Laboratories A300-997A), SDE2 (Bethyl Laboratories A302-098A and A302-099A), GAPDH (Santa Cruz Biotechnologies sc-47724), U2AF1 (Bethyl Laboratories A302-079A), CUL7 (Bethyl Laboratories A300-223A), PITPNM1 (Abcam AB254959), Puromycin (EMD Millipore MABE343), Histone 4 (Active Motif #39269), eIF2α (Cell Signaling Technology 9722S), Phospho- eIF2α−Ser51 (Cell Signaling Technology 9721S), 4E-BP1 (Cell Signaling Technology 9644S), Phospho-4E-BP1-Thr37/46 (Cell Signaling Technology 2855S), Cactin (Bethyl Laboratories A303-349A), FBL (Abcam ab5821), Tubulin (Cell Signaling Technology 2125S), p54 (Santa Cruz Biotechnology sc-126) and p21 (Santa Cruz Biotechnology sc-6246). The following plasmids were used: pLKO.1.
Floro J., Dai A., Metzger A., Mora-Martin A., Ganem N.J., Cifuentes D., Wu C.S., Dalal J., Lyons S.M., Labadorf A, & Flynn R.L. (2021). SDE2 is an essential gene required for ribosome biogenesis and the regulation of alternative splicing. Nucleic Acids Research, 49(16), 9424-9443.
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