antibodies against u2af2  (Novus Biologicals)

Journal: Heliyon

Article Title: LncRNA CECR7 boosts hepatocellular carcinoma progression by recruiting RNA binding protein U2AF2 to enhance the stability of EXO1 mRNA

doi: 10.1016/j.heliyon.2023.e19862

Figure Lengend Snippet: CECR7 stabilize EXO1 mRNA under the mediation of U2AF2 protein. (A) The platform starBase ( http://starbase.sysu.edu.cn ) was applied to predict the potential RNA binding protein for both CECR7 and EXO1 mRNA. ( B, C ) RIP assay using antibody against U2AF2 to determine the enrichment of CECR7 and EXO1 mRNA in HCC cells (Mean ± SD; n = 3). ** P < 0.01, *** P < 0.001, Student's t -test. ( D, E ) RT-qPCR analysis and Western blot were used to detect the effect of CECR7 on U2AF2 expression. ( F, G ) U2AF2-overexpressing reversed the repression of EXO1 mRNA by CECR7 shRNA#1, and the EXO1 mRNA induction induced by CECR7-overexpressing was abrogated by U2AF2 shRNA (Mean ± SD; n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA.

Article Snippet: Lysates of HCC cells were cultured in RIP containing magnetic beads conjugated with antibodies against U2AF2 (NBP2-33397, Novus, USA) or control IgG (NBP2-34250, Novus, USA) for 6h at 4 °C.

Techniques: RNA Binding Assay, Quantitative RT-PCR, Western Blot, Expressing, shRNA

Journal: Molecular Cancer

Article Title: HPGD induces ferroptosis and autophagy to suppress esophageal squamous cell carcinoma through the LXA4–ERK1/2–U2AF2–TFRC axis

doi: 10.1186/s12943-025-02464-x

Figure Lengend Snippet: ༎HPGD promotes the binding of RNA-binding protein (RBP) U2AF2 to the TFRC promoter, which improves its transcription by suppressing ERK1/2 signaling activation. (A) Construction of truncated TFRC promoter plasmids and dual-luciferase reporter assay analysis of the TFRC promoter region after transfection into 293 T cells. (B) Biotin labeling of the TFRC promoter region obtained from the dual-luciferase reporter assay, followed by Lipofectamine transfection into 293T cells, ultrasonic lysis, biotin pulldown, and SDS-PAGE to analyze differential proteins via MS-MS. (C) Biotinylated promoter transfected into 293 T cells, followed by biotin pulldown and Western blot validation of promoter-binding proteins. (D) ChIP assay to validate U2AF2 binding to the TFRC promoter in ESCC cells. (E) A dual luciferase plasmid with a mutated U2AF2 binding site in the TFRC promoter was co-transfected with a U2AF2 overexpression plasmid into 293 T cells to measure luciferase activity. (F) RNA-RIP experiments were performed utilizing U2AF2 and IgG antibodies. Following the purification of RNA and protein, TFRC was detected through PCR, while U2AF2 was identified using immunoblotting. G-H. Western blot analysis of U2AF2 expression levels in cells after HPGD overexpression. I. ChIP assay analysis of TFRC promoter activity after treatment with the Erk1/2 activator EGF in control and HPGD overexpressing cells. J. FISH analysis with TFRC promoter probes and U2AF2 antibodies to detect colocalization. K-L. Western blot detection of interference efficiency after silencing U2AF2 with small interfering RNA. M-N. Western blot detection of TFRC expression after silencing U2AF2 in ESCC cells with HPGD overexpression. *: P < 0.05,** P < 0.01,*** P < 0.001, **** P < 0.0001

Article Snippet: Chromatin immunoprecipitation was conducted overnight at 4 °C with primary antibodies against U2AF2 (Proteintech, America) and 2 μl of normal rabbit IgG (provided in the ChIP Assay Kit).

Techniques: Binding Assay, RNA Binding Assay, Activation Assay, Luciferase, Reporter Assay, Transfection, Labeling, Lysis, SDS Page, Tandem Mass Spectroscopy, Western Blot, Biomarker Discovery, Plasmid Preparation, Over Expression, Activity Assay, Purification, Expressing, Control, Small Interfering RNA

Journal: Regenerative Therapy

Article Title: Exosomal U2AF2 derived from human bone marrow mesenchymal stem cells attenuates the intervertebral disc degeneration through circ_0036763/miR-583/ACAN axis

doi: 10.1016/j.reth.2024.01.006

Figure Lengend Snippet: U2AF2/circ_0036763/miR-583/ACAN axis is associated with IDD development . (A) The expression of circ_0036763 in HNPCs isolated from IDD patients and normal HNPCs was evaluated by qRT-PCR. (B) The putative interaction between circ_0036763 and miR583 was predicted by bioinformatic analysis. (C) The expression of miR583 in HNPCs isolated from IDD patients and normal HNPCs was evaluated by qRT-PCR. (D) The expression of U2AF2 in HNPCs isolated from IDD patients and normal HNPCs was evaluated by WB. (E) The putative interaction between miR-583 and ACAN was predicted by bioinformatic analysis. (F) The expression of ACAN in HNPCs isolated from IDD patients and normal HNPCs was evaluated by Western blot. (G) HNPCs isolated from IDD patients were transfected with circ_0036763 OE or the empty vector (vec), and the expression of ACAN, Collagen I and Collagen II was evaluated by WB. ∗ P < 0.05, ∗∗ P < 0.01 and ns indicates no significant difference.

Article Snippet: After blocking by 5 % skim milk for 1 h at RT, the membranes were incubated with primary antibody against U2AF2 (1:1000, #15624-1-AP, Proteintech, China), ACAN (1:1000, #bs-1223 R, Bioss, China), Collagen I (1:1000, #bs-7158 R, Bioss), Collagen II (1:1000, #bs-10589 R, Bioss), CD63 (1:1000, #bs-1523 R, Bioss), CD81 (1:1000, #bs-6934 R, Bioss) or internal reference GAPDH (1:10000, #KC-5G5, Aksomics, China) overnight at 4 °C, respectively.

Techniques: Expressing, Isolation, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation

Journal: Regenerative Therapy

Article Title: Exosomal U2AF2 derived from human bone marrow mesenchymal stem cells attenuates the intervertebral disc degeneration through circ_0036763/miR-583/ACAN axis

doi: 10.1016/j.reth.2024.01.006

Figure Lengend Snippet: The RBP-circRNA interaction between circ_0036763 and U2AF2.

Article Snippet: After blocking by 5 % skim milk for 1 h at RT, the membranes were incubated with primary antibody against U2AF2 (1:1000, #15624-1-AP, Proteintech, China), ACAN (1:1000, #bs-1223 R, Bioss, China), Collagen I (1:1000, #bs-7158 R, Bioss), Collagen II (1:1000, #bs-10589 R, Bioss), CD63 (1:1000, #bs-1523 R, Bioss), CD81 (1:1000, #bs-6934 R, Bioss) or internal reference GAPDH (1:10000, #KC-5G5, Aksomics, China) overnight at 4 °C, respectively.

Techniques:

Journal: Regenerative Therapy

Article Title: Exosomal U2AF2 derived from human bone marrow mesenchymal stem cells attenuates the intervertebral disc degeneration through circ_0036763/miR-583/ACAN axis

doi: 10.1016/j.reth.2024.01.006

Figure Lengend Snippet: U2AF2 promotes the mature of circ_0036763 . (A) The direct interaction between pre-circ_0036763 and U2AF2 was determined by RIP assay. (B) The three siRNAs targeting U2AF2 were transfected into HNPCs, and the transfection efficiency was confirmed by qRT-PCR. (C) The overexpressing plasmid for U2AF2 (U2AF2 OE) and si-U2AF2 were transfected into HNPCs, and the expression of circ_0036763 was evaluated by qRT-PCR. (D) The overexpressing plasmid for U2AF2 (U2AF2 OE) was transfected into HNPCs isolated from IDD patients, and the expression of circ_0036763 was evaluated by qRT-PCR. (E) The level of pre-circ_0036763 in HNPCs isolated from IDD patients and normal HNPCs detected by qRT-PCR. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 and ns indicates no significant difference.

Article Snippet: After blocking by 5 % skim milk for 1 h at RT, the membranes were incubated with primary antibody against U2AF2 (1:1000, #15624-1-AP, Proteintech, China), ACAN (1:1000, #bs-1223 R, Bioss, China), Collagen I (1:1000, #bs-7158 R, Bioss), Collagen II (1:1000, #bs-10589 R, Bioss), CD63 (1:1000, #bs-1523 R, Bioss), CD81 (1:1000, #bs-6934 R, Bioss) or internal reference GAPDH (1:10000, #KC-5G5, Aksomics, China) overnight at 4 °C, respectively.

Techniques: Transfection, Quantitative RT-PCR, Plasmid Preparation, Expressing, Isolation

Journal: Regenerative Therapy

Article Title: Exosomal U2AF2 derived from human bone marrow mesenchymal stem cells attenuates the intervertebral disc degeneration through circ_0036763/miR-583/ACAN axis

doi: 10.1016/j.reth.2024.01.006

Figure Lengend Snippet: U2AF2 from bMSCs-derived exosomes regulates expressions of circ_0036763 and ACAN in HNPCs isolated from IDD patients . (A and B) The level of U2AF2 in HNPCs and bMSCs-derived exosomes was evaluated by qRT-PCR (A) and WB (B). (C) BMSCs or bMSCs-derived exosomes were co-cultured with HNPCs isolated from IDD patients. When needed, exosomes inhibitor G4869 was added. The expression of U2AF2 in HNPCs isolated from IDD patients was evaluated by WB. (D and E) BMSCs-derived exosomes, or si-U2AF2 transfected bMSCs were co-cultured with HNPCs isolated from IDD patients. (D) The level of circ_0036763 in HNPCs isolated from IDD patients was evaluated by qRT-PCR. (E) The expression of ACAN in HNPCs isolated from IDD patients was evaluated by Western blot. ∗∗ P < 0.01.

Article Snippet: After blocking by 5 % skim milk for 1 h at RT, the membranes were incubated with primary antibody against U2AF2 (1:1000, #15624-1-AP, Proteintech, China), ACAN (1:1000, #bs-1223 R, Bioss, China), Collagen I (1:1000, #bs-7158 R, Bioss), Collagen II (1:1000, #bs-10589 R, Bioss), CD63 (1:1000, #bs-1523 R, Bioss), CD81 (1:1000, #bs-6934 R, Bioss) or internal reference GAPDH (1:10000, #KC-5G5, Aksomics, China) overnight at 4 °C, respectively.

Techniques: Derivative Assay, Isolation, Quantitative RT-PCR, Cell Culture, Expressing, Transfection, Western Blot

Journal: Heliyon

Article Title: LncRNA CECR7 boosts hepatocellular carcinoma progression by recruiting RNA binding protein U2AF2 to enhance the stability of EXO1 mRNA

doi: 10.1016/j.heliyon.2023.e19862

Figure Lengend Snippet: CECR7 stabilize EXO1 mRNA under the mediation of U2AF2 protein. (A) The platform starBase ( http://starbase.sysu.edu.cn ) was applied to predict the potential RNA binding protein for both CECR7 and EXO1 mRNA. ( B, C ) RIP assay using antibody against U2AF2 to determine the enrichment of CECR7 and EXO1 mRNA in HCC cells (Mean ± SD; n = 3). ** P < 0.01, *** P < 0.001, Student's t -test. ( D, E ) RT-qPCR analysis and Western blot were used to detect the effect of CECR7 on U2AF2 expression. ( F, G ) U2AF2-overexpressing reversed the repression of EXO1 mRNA by CECR7 shRNA#1, and the EXO1 mRNA induction induced by CECR7-overexpressing was abrogated by U2AF2 shRNA (Mean ± SD; n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA.

Article Snippet: The protein lysates were incubated with primary antibody against U2AF2 (1:1000, NBP2-33397, Novus, USA), EXO1 (1:1000, NBP2-16391, Novus, USA) or β-actin (1:1000, ab8226, Abcam, USA).

Techniques: RNA Binding Assay, Quantitative RT-PCR, Western Blot, Expressing, shRNA