Anti-UFC1 antibody [EPR15014-102]

8 product images

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  Western blot - Anti-UFC1 antibody [EPR15014-102] (ab189252)

  Lanes 1- 2: Merged signal (red and green). Green - ab189252 observed at 20 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.
  

  ab189252 was shown to react with UFC1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line Human UFC1 knockout HEK-293T cell line ab266814 (knockout cell lysate Human UFC1 knockout HEK-293T cell lysate ab257781) was used. Wild-type HEK-293T and UFC1 HEK-293T KO cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab189252 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-UFC1 antibody [EPR15014-102] (ab189252) at 1/1000 dilution

  Lane 1: Wild-type HEK-293T cell lysate at 20 µg

  Lane 2: UFC1 knockout HEK-293T cell lysate at 20 µg

  Lane 2: Western blot - Human UFC1 knockout HEK-293T cell line (Human UFC1 knockout HEK-293T cell line ab266814)

  Performed under reducing conditions.

  Predicted band size: 19 kDa

  Observed band size: 20 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-UFC1 antibody [EPR15014-102] (ab189252)

  Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human colon tissue labeling UFC1 with ab189252 at 1/100 dilution followed by pre-diluted HRP Polymer for Rabbit IgG secondary antibody and counter-stained with Hematoxylin.
  

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Western blot - Anti-UFC1 antibody [EPR15014-102] (ab189252)

  Lanes 1 - 4: Merged signal (red and green). Green - ab189252 observed at 20 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
  

  ab189252 was shown to react with UFC1 in wild-type HAP1 cells in western blot. Loss of signal was observed when UFC1 knockout sample was used. Wild-type HAP1 and UFC1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab189252 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-UFC1 antibody [EPR15014-102] (ab189252) at 1/10000 dilution

  Lane 1: Wild-type HAP1 cell lysate at 20 µg

  Lane 2: UFC1 knockout HAP1 cell lysate at 20 µg

  Lane 3: MCF7 cell lysate at 20 µg

  Lane 4: A549 cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 19 kDa

  Observed band size: 20 kDa

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  Western blot - Anti-UFC1 antibody [EPR15014-102] (ab189252)

  All lanes: Western blot - Anti-UFC1 antibody [EPR15014-102] (ab189252) at 1/20000 dilution

  Lane 1: Human fetal liver lysate at 20 µg

  Lane 2: U87-MG cell lysate at 20 µg

  Lane 3: MCF7 cell lysate at 20 µg

  Lane 4: A549 cell lysate at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

  Predicted band size: 19 kDa

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  Western blot - Anti-UFC1 antibody [EPR15014-102] (ab189252)

  All lanes: Western blot - Anti-UFC1 antibody [EPR15014-102] (ab189252) at 1/5000 dilution

  Lane 1: RAW 264.7 cell lysate at 10 µg

  Lane 2: NIH 3T3 cell lysate at 10 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

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  Immunoprecipitation - Anti-UFC1 antibody [EPR15014-102] (ab189252)

  Western blot analysis of immunoprecipitation pellet from MCF7 cell lysate immunoprecipitated using ab189252 at 1/50 dilution (lane 1) or PBS control (lane 2).
  Secondary: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1500 dilution.

  All lanes: Immunoprecipitation - Anti-UFC1 antibody [EPR15014-102] (ab189252)

  Predicted band size: 19 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-UFC1 antibody [EPR15014-102] (ab189252)

  Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human transitional cell carcinoma of bladder tissue labeling UFC1 with ab189252 at 1/100 dilution followed by pre-diluted HRP Polymer for Rabbit IgG secondary antibody and counter-stained with Hematoxylin.
  

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Image collected and cropped by CiteAb under a CC-BY license from the publication

  Western blot - Anti-UFC1 antibody [EPR15014-102] (ab189252)

  UFC1 western blot using anti-UFC1 antibody [EPR15014-102] ab189252. Publication image and figure legend from Qin, B., Yu, J., et al., 2019, Nat Commun, PubMed 30886146.

  
  

  ab189252 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab189252 please see the product overview.

  UFL1 monoufmylates histone H4 and promotes ATM activation. a Selected proteins identified by mass spectrometry from irradiated 293T cell expressing Flag-His vector or Flag-His-UFM1. N = 1 sample in each group was analyzed. The full list of identified proteins is provided in Supplementary Data 1. Among histone proteins, only H4 is enriched in the Flag-His-UFM1 purification (9 unique/20 total peptides) compared to the Flag-His purification (6 unique/7 total peptides), suggesting that H4 might be ufmylated. b Flag-His-ufmylated proteins were purified from 293T cells before and after IR (2 Gy) after purification with nickel beads and anti-Flag agarose. The immunoprecipitates were detected with indicated antibodies. c Flag-His-ufmylated H4 was purified from control and UFL1 knockdown cells with or without 2 Gy IR and blotted with indicated antibodies. d In vitro ufmylation assay. Purified UBA5, UFC1, UFL1, UFM1, and H4 proteins were incubated together in the presence of ATP and MgCl2 at 30 °C for 90 min. The reaction products were probed with indicated antibodies. e Wildtype (WT) histone H4 and 11 different single lysine (K) to arginine (R) mutants were transfected into U2OS cells. Flag and His tandem purification was performed and H4 ufmylation was analyzed. f Constructs expressing WT or K31R H4 were transfected into U2OS tet-on UFL1 shRNA expressing cells, and the cells were treated with doxycycline as indicated. Thirty minutes after 2 Gy IR, the cells were harvested and blotted with indicated antibodies. g Colony formation of U2OS cells expressing WT H4 or H4K31R following IR. The data presented are mean ± s.e.m. for n = 3 independent experiments. Statistical significance was calculated using two-way ANOVA. *p < 0.05, **p < 0.01. h U2OS cells stably expressing UFL1 Tet-on shRNA were treated with doxycycline (Dox) and irradiated with 2 Gy IR. Thirty minute later, cells were harvested. Half of the cells were lysed. Chromatin fractions were isolated from other half of cells. The samples were blotted with indicated antibodies. Source data are provided as a Source Data file