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U2af65

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U2AF65 is a protein involved in pre-mRNA splicing. It recognizes the polypyrimidine tract near the 3' splice site and helps recruit the U2 snRNP to the pre-mRNA.

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Lab products found in correlation

Flag m2, by Merck Group (3 mentions) Hnrnp f h, by Abcam (2 mentions) β actin, by Merck Group (2 mentions) Srrm1, by Abcam (2 mentions) Srsf1, by Santa Cruz Biotechnology (2 mentions) Srsf2, by Abcam (2 mentions) Alexa fluor 488 conjugated goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions) α tubulin, by Merck Group (1 mentions) Cfim59, by Fortis Life Sciences (1 mentions) Cfim68, by Abcam (1 mentions) Chemidoc mp system, by Bio-Rad (1 mentions) U2af2, by Santa Cruz Biotechnology (1 mentions) Tasor, by Merck Group (1 mentions) Srrm2, by Merck Group (1 mentions) Hnrnpa1, by Santa Cruz Biotechnology (1 mentions) Hnrnpc1 c2, by Santa Cruz Biotechnology (1 mentions) M igg, by Cell Signaling Technology (1 mentions) H3k27ac, by Vector Laboratories (1 mentions) H3k9ac, by Vector Laboratories (1 mentions) Lamin a c, by Santa Cruz Biotechnology (1 mentions)

Close spelling variants of this product

Anti-U2AF65 U2AF65 (MC3,

9 protocols using u2af65

1

Whole-Mount Immunostaining in Locust Brain

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Whole‐mount double immunohistochemistry in the locust brain was performed by using affinity‐purified monoclonal mouse antibodies against Piwi1, Piwi2, and Ago3 (1:100, BGI), hnRNP F/H (1:100, Abcam), and U2AF65 (1:50, Santa Cruz Biotechnology). An Alexa Fluor 488‐conjugated goat anti‐mouse antibody (1:500, Life Technologies Cat. A‐11008) was used as the secondary antibody for Piwi1, Piwi2, and Ago3 staining. For lipid staining, lipids were visualized by staining the locust fat body with Nile red (0.5 mg/ml, Thermo Fisher N‐1142) for 1 h at room temperature.
Wang H., Jiang F., Liu X., Liu Q., Fu Y., Li R., Hou L., Zhang J., He J, & Kang L. (2022). Piwi/piRNAs control food intake by promoting neuropeptide F expression in locusts. EMBO Reports, 23(3), e50851.
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2

Protein Extraction and Immunoblotting Protocol

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Protein extracts were prepared by lysis in Laemmli buffer [62.5 mM Tris-HCl (pH 6.8), 5% 2-mercaptoethanol, 10% glycerol, 2% SDS, 0.002% bromophenol blue]. Blots were probed with the following antibodies: U2AF65 (Santa Cruz Biotechnology, MC3), MYC (Roche, 11-667-149-001), β-actin (Sigma, AC74), FLAG M2 (Sigma, F1804), ATG7 (ProSci, 3615), CFIm59 (Bethyl Laboratories, A301-359A), CFIm68 (Abcam, ab175237), LC3B (Cell Signaling Technology, #3868), p62 (Santa Cruz Biotechnology, sc-28359), U2AF35 (Proteintech, 10334-1-AP) and α-tubulin (Sigma, B5-1-2). Immunoblots were visualized by a ChemiDoc™ MP System (Bio-Rad).
Park S.M., Ou J., Chamberlain L., Simone T.M., Yang H., Virbasius C.M., Ali A.M., Zhu L.J., Mukherjee S., Raza A, & Green M.R. (2016). U2AF35(S34F) Promotes Transformation by Directing Aberrant ATG7 Pre-mRNA 3’ End Formation. Molecular cell, 62(4), 479-490.
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3

Comprehensive Antibody Panel for Splicing Analysis

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The following antibodies were used: AFB1 (Santa Cruz, no. sc-393403), SAFB2 (Santa Cruz, no. sc-514963), SAFB1/2 (HET) (human: Merck/Sigma-Aldrich, no. sc05-588; mouse: LSBio, no. LS-C2886411), SLTM (Invitrogen, no. PA5-59154), ORF1p (human: abcam, no. ab230966; mouse: abcam, no. ab216324), TASOR (Sigma-Aldrich, no. HPA006735), 1H4 (p-SR) (Merck/Sigma-Aldrich, no. MABE50), RBM12B (Bethyl, no. A305-871A-T), RBMX (Cell Signaling Technology, no. 14794 S), NCOA5 (Bethyl, no. A300-790A-T), ZNF638 (Sigma-Aldrich, no. ZRB1186), ZNF326 (Santa Cruz, no. sc-390606), TRA2B (Bethyl, no. A305-011A-M), U2AF2 (U2AF65; Santa Cruz, no. sc-53942), TUBULIN (Santa Cruz, no. sc-32293), SRRM1 (abcam, no. ab221061), SRRM2 (SC35) (Sigma-Aldrich, no. S4045), SON (Sigma-Aldrich, no. HPA023535), DHX9 (abcam, no. ab183731), U1-70K (SySy, no. 203011), PRP8 (Santa Cruz, no. sc-55533), RNAPII (Creative Biolabs, no. CBMAB-XB0938-YC), IgG normal mouse (Santa Cruz, no. sc-2025), SRSF1 (Santa Cruz, no. sc-33652), SRSF2 (abcam, no. ab204916), SRSF3 (Elabscience, no. E-AB-32966), SRSF7 (MBL, no. RN079PW), RB1 (Cell Signaling Technology, no. 9309 S), TRA2B (Santa Cruz, no. sc-166829) and YTHDC1 (Proteintech, no. 14392-1-AP).
Ilık İ.A., Glažar P., Tse K., Brändl B., Meierhofer D., Müller F.J., Smith Z.D, & Aktaş T. (2024). Autonomous transposons tune their sequences to ensure somatic suppression. Nature, 626(8001), 1116-1124.
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4

Comprehensive Antibody Panel for Epigenetics

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R-IgG (SC-2027), m-IgG (SC-2025), Actin (SC-47778), H3K9me3 (Cell Signaling, 9754), H3K4me3 (Cell Signaling, 9751; active motif 39159), H3K27me3 (Cell Signaling, 9733), H3K9ac (Cell Signaling, 9649), H3K36me (Cell Signaling, 4909), H3K27ac (ab4729), H3K9ac (ab176916), rabbit polyclonal Ki67 (Vectorlabs), MLL1 (Active motif, 61296), Lamin A/C (E-1), hnRNPC1/C2 (Santa Cruz, SC-32308), SAFA (Santa Cruz, SC-32315), U2AF65 (Santa Cruz, SC-53942), DDX3 (Santa Cruz, SC-365768), hnRNPA1 (Santa Cruz, SC-32301), hnRNPD (abcam, ab61193), DDX21 (Santa Cruz, SC-376953), DNA Damage antibody sample kit (Cell Signaling, 9947), Apoptosis Antibody sampler Kit (Cell signaling, 9915).
Puvvula P.K, & Moon A.M. (2021). Novel Cell-Penetrating Peptides Derived From Scaffold-Attachment- Factor A Inhibits Cancer Cell Proliferation and Survival. Frontiers in Oncology, 11, 621825.
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5

Locust Brain Protein Analysis

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Locust brains (8–10 individuals/sample) were collected and homogenized in lysis buffer (CWBIO) containing protease inhibitor (CWBIO). The total protein content was determined using a bicinchoninic acid protein assay kit (Thermo Scientific). The extracts were reduced, denatured, and separated by gel electrophoresis on a 10% SDS‐PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated separately with specific antibodies against Piwi1, Piwi2, and Ago3 (mouse anti‐Piwi1 serum, 1:500; mouse anti‐Piwi2 serum, 1:500; and mouse anti‐Ago3 serum, 1:500, respectively), hnRNP F/H (Mouse monoclonal antibody, 1:2,000, Abcam), and U2AF65 (Mouse monoclonal antibody, 1:200, Santa Cruz Biotechnology). Tubulin was used as an endogenous control (rabbit polyclonal antibody, 1:5,000, CWBIO). Goat anti‐rabbit IgG was used as the secondary antibody (1:10,000, CWBIO). The intensities of the Western blot bands were quantified using densitometry with Quantity One software.
Wang H., Jiang F., Liu X., Liu Q., Fu Y., Li R., Hou L., Zhang J., He J, & Kang L. (2022). Piwi/piRNAs control food intake by promoting neuropeptide F expression in locusts. EMBO Reports, 23(3), e50851.
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6

Characterization of Prostate Cancer Cell Lines

Cited 2 times
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LNCaP, CWR22Rv1 and VCaP cells were obtained in 2001 from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured in RPMI containing 10% complete FBS and penicillin/streptomycin. ATCC uses Short Tandem Repeat (STR) profiling for testing and authentication of cell lines. C4-2B cells were kindly provided and authenticated by Dr. Leland Chung, Cedars-Sinai Medical Center, Los Angeles, CA in 2006. All experiments with these cell lines were performed within 6 months of resuscitation after cryopreservation. LNCaP cells stably expressing NF-kappaB2/p52 were generated by stable transfection of LNCaP cells with plasmids expressing NF-kappaB2/p52 as described previously (21 (link)) and were not authenticated further. 22Rv1 and C4-2B cells resistant to enzalutamide (22Rv1-Enza-R and C4-2B-Enza-R) were generated by chronic culture of 22Rv1 and C4-2B cells in enzalutamide as described previously (22 (link), 23 (link)) and were not authenticated further. Antibodies against NF-kappaB2/p52 (K-27), AR (441; mouse monoclonal), HA, Tubulin, U2AF65 and ASF/SF2 were from Santa Cruz Biotechnologies. Antibodies against splicing factors hnRNPA1 (9H10) and hnRNPA2B1 (DP3B3) were from Sigma-Aldrich and AbCam respectively. Sso Fast™ Eva Green qPCR Supermix was from Bio-Rad. All other reagents were of analytical grade and obtained from local suppliers.
Nadiminty N., Tummala R., Liu C., Lou W., Evans C.P, & Gao A.C. (2015). NF-kappaB2/p52:c-Myc:hnRNPA1 pathway regulates expression of androgen receptor splice variants and enzalutamide sensitivity in prostate cancer. Molecular cancer therapeutics, 14(8), 1884-1895.
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7

Comprehensive Immunoblotting Antibody Panel

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ADAR1(Santa Cruz Biotechnology, sc-73408), alpha-Tubulin (Santa Cruz Biotechnology, sc-32293), COIL (Cell Signaling Technology, D2L3J, #14168), DHX9 (Abcam, ab183731), FLAG-M2 (Sigma, F3165), GFP (Chromotek, PAGB1), Myc-tag (Cell Signaling Technology, 9B11, #2276), Phospho-Rpb1 CTD (Ser2) (Cell Signaling Technology, E1Z3G, #13499), PNN (Abcam, ab244250), RBM25 (Sigma, HPA070713-100UL), SC-35 (Sigma, S4045), SRSF1 (SF2/ASF, Santa Cruz Biotechnology, sc-33652), SON (polyclonal rabbit, Sigma, HPA023535), SRRM1 (Abcam, ab221061), SRRM2 (Thermo Fisher Scientific, PA5-66827), SRSF2 (Abcam, ab28428), SRSF7 (MBL, RN079PW), U1-70K (Santa Cruz Biotechnology, sc-390899), U2AF65 (Santa Cruz Biotechnology, sc-53942).
Ilik İ.A., Malszycki M., Lübke A.K., Schade C., Meierhofer D, & Aktaş T. (2020). SON and SRRM2 are essential for nuclear speckle formation. eLife, 9, e60579.
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8

Affinity Purification of CFIm59/68

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HEK293T cells were transfected with a construct expressing Flag-CFIm59 or Flag-CFIm68, and 56 h later cells were harvested, lysed and briefly sonicated. Extracts were cleared by centrifugation and incubated with anti-FLAG M2 affinity gel (Sigma-Aldrich). Beads were washed three times, treated with 10 μg/ml RNase A, and washed again. Flag-CFIm59/68-bound beads were incubated with Ba/F3-U2AF35 or Ba/F3-U2AF35(S34F) nuclear extracts prepared as described in Supplemental Experimental Procedures. Proteins were eluted and analyzed by immunoblotting with the following antibodies: U2AF65 (Santa Cruz Biotechnology, MC3), MYC (Roche, 11-667-149-001), β-actin (Sigma, AC74), FLAG M2 (Sigma, F1804).
Park S.M., Ou J., Chamberlain L., Simone T.M., Yang H., Virbasius C.M., Ali A.M., Zhu L.J., Mukherjee S., Raza A, & Green M.R. (2016). U2AF35(S34F) Promotes Transformation by Directing Aberrant ATG7 Pre-mRNA 3’ End Formation. Molecular cell, 62(4), 479-490.
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9

Protein Extraction and Immunoblotting Procedure

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Cells were lysed with a lysis buffer (20 mM Tris [pH 7.5], 0.1% Triton X-100, 0.5% deoxycholate, 1 mM PMSF, 10 μg/ml aprotinin, and 10 μg/ml leupeptin) and then cleared via centrifugation at 4°C. The total protein concentration was determined using a Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions, and the obtained proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transferring the proteins onto nitrocellulose membranes and blocking the membranes, the membranes were incubated overnight at 4°C with antibodies in phosphate-buffered saline containing 0.1% Tween 20. The primary antibodies employed for immunoblotting were as follows: anti-β-actin antibody (Santa Cruz Biotechnology), anti-KRT1 antibody (Abcam), anti-KRT10 antibody (Covance, Richmond, VA, USA), anti-p53 antibody (Wako), p44/42 ERK antibody (Cell Signaling Inc., Beverly, MA, USA), phospho-p44/42 ERK antibody (Cell Signaling Inc.), U170K antibody (Santa Cruz Biotechnology), U2AF65 (Santa Cruz Biotechnology), PRP8 antibody (Santa Cruz Biotechnology), FGF2 antibody (Santa Cruz Biotechnology), and NRAS antibody (Santa Cruz Biotechnology). All protein bands were detected using an ECL system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
Kim K.H., Cho E.G., Yu S.J., Kang H., Kim Y.J., Kim S.H, & Lee T.R. (2015). ΔNp63 intronic miR-944 is implicated in the ΔNp63-mediated induction of epidermal differentiation. Nucleic Acids Research, 43(15), 7462-7479.
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