- MA1-199 - Invitrogen Antibodies HNF4A antibody

Submitted references SARS-CoV-2 infection of human pluripotent stem cell-derived liver organoids reveals potential mechanisms of liver pathology.

Richards A, Friesen M, Khalil A, Barrasa MI, Gehrke L, Jaenisch R
iScience 2022 Oct 21;25(10):105146

In vitro grafting of hepatic spheroids and organoids on a microfluidic vascular bed.

Bonanini F, Kurek D, Previdi S, Nicolas A, Hendriks D, de Ruiter S, Meyer M, Clapés Cabrer M, Dinkelberg R, García SB, Kramer B, Olivier T, Hu H, López-Iglesias C, Schavemaker F, Walinga E, Dutta D, Queiroz K, Domansky K, Ronden B, Joore J, Lanz HL, Peters PJ, Trietsch SJ, Clevers H, Vulto P
Angiogenesis 2022 Nov;25(4):455-470

DevKidCC allows for robust classification and direct comparisons of kidney organoid datasets.

Wilson SB, Howden SE, Vanslambrouck JM, Dorison A, Alquicira-Hernandez J, Powell JE, Little MH
Genome medicine 2022 Feb 22;14(1):19

Establishment of Human Leukocyte Antigen-Mismatched Immune Responses after Transplantation of Human Liver Bud in Humanized Mouse Models.

Mori A, Murata S, Tashiro N, Tadokoro T, Okamoto S, Otsuka R, Wada H, Murata T, Takahashi T, Seino KI, Taniguchi H
Cells 2021 Feb 23;10(2)

Enhanced expression of HNF4α during intestinal epithelial differentiation is involved in the activation of ER stress.

Tunçer S, Sade-Memişoğlu A, Keşküş AG, Sheraj I, Güner G, Akyol A, Banerjee S
The FEBS journal 2020 Jun;287(12):2504-2523

An Unexpected Role of Cholesterol Sulfotransferase and its Regulation in Sensitizing Mice to Acetaminophen-Induced Liver Injury.

An Y, Wang P, Xu P, Tung HC, Xie Y, Kirisci L, Xu M, Ren S, Tian X, Ma X, Xie W
Molecular pharmacology 2019 Jun;95(6):597-605

Regulation of Cholesterol Sulfotransferase SULT2B1b by Hepatocyte Nuclear Factor 4α Constitutes a Negative Feedback Control of Hepatic Gluconeogenesis.

Bi Y, Shi X, Zhu J, Guan X, Garbacz WG, Huang Y, Gao L, Yan J, Xu M, Ren S, Ren S, Liu Y, Ma X, Li S, Xie W
Molecular and cellular biology 2018 Apr 1;38(7)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of HNF4 alpha was performed by loading 20 µg of human hepatoblast and HepG2 whole cell lysate and 10 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) onto a 4-20% Tris-Glycine polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 5% Milk in TBST for at least 1 hour at room temperature. HNF4 alpha was detected at ~53 kDa using a HNF4 alpha mouse monoclonal antibody (Product # MA1-199) at a dilution of 1 µg/mL in 5% Milk in TBST overnight at 4C on a rocking platform, followed by a Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 31430) at a dilution of 1:10,000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal Pico substrate (Product # 34078) and the myECL Imager (Product # 62236).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-HNF4A Monoclonal Antibody (K9218) (Product # MA1-199) and a 53kDa band corresponding to HNF4A was observed across all the cell lines tested expect HCT-116 and HeLa. Whole cell extracts (30 µg lysate) of Caco-2 (Lane 1), HCT 116 (Lane 2), Hep G2 (Lane 3) and HeLa (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of HNF4 alpha (green) in HepG2 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes, and blocked with 0.3% BSA in PBS (Product # 37525) for 30 minutes at room temperature. Cells were stained with a HNF4 alpha mouse monoclonal antibody (Product # MA1-199) at a dilution of 2.5 µg/mL in staining buffer for 1 hour at room temperature, and then incubated with a Goat anti-Mouse IgG Secondary Antibody, DyLight 488 conjugate (Product # 35503) at a dilution of 1:500 for 1 hour at room temperature (green). Nuclei (blue) were counterstained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of HNF4A was performed using 70% confluent log phase HepG2 and HeLa cells. The cells were fixed with 4% Paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 10 minutes at room temperature. The cells were labeled with Anti-HNF4A Monoclonal Antibody (Product # MA1-199) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175, 1:2000 dilution) for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents HeLa cells having no expression of HNF4A. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of HNF4A was performed in iPSC derived liver organoid. The cells were fixed with 4% Paraformaldehyde for 30 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Anti-HNF4A Monoclonal Antibody (Product # MA1-199) at 1:100 dilution in 0.1% BSA, incubated at 4°C overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175, 1:2,000 dilution) for 45 minutes at room temperature. The panels represent the merged images of HNF4A Monoclonal Antibody (green) and DAPI (blue) staining for iPSC control cells and differentiated liver organoid. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of HNF4 alpha was done on HepG2 cells. Cells were fixed, permeabilized and stained with a HNF4 alpha mouse monoclonal antibody (Product # MA1-199, red histogram) or a mouse IgG2a isotype control (Product # MA1-10418, black histogram) at a dilution of 10 µg/mL. After incubation for 1 hour on ice, the cells were labeled with a Goat anti-Mouse IgG Secondary Antibody, DyLight 650 conjugate (Product # 84545) at a dilution of 1:50 for 1 hour on ice. A representative 10,000 cells were acquired and analyzed for each sample.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: FIG 7 Thiocholesterol (TC) shows an improved intracellular stability and better efficacy in inhibiting gluconeogenesis in primary hepatocytes. (A) Schematic depiction of the synthesis of TC from cholesteryl chloride. (B) Mouse primary hepatocytes isolated from 8-week-old male WT mice were treated with 5 muM TC or CS for up to 48 h, and the intracellular concentrations of TC and CS were measured by using UPLC-mass spectrometry. (C and D) Glucose production (C) and expression of gluconeogenic genes (D) in mouse primary hepatocytes treated with the indicated concentrations of drugs for 24 h. Cells were treated with 10 muM FSK for 2.5 h before the glucose production assay. The expression of each gene or glucose production was arbitrarily set as 1 in cells treated with DMSO. (E and F) The designs of the experiments were similar to those described for panels C and D, except that human primary hepatocytes were used. (G) The subcellular distribution of Hnf4alpha was visualized by immunofluorescence using an anti-Hnf4alpha antibody (red) in primary hepatocytes isolated from WT mice and treated with the indicated concentrations of drugs. DAPI (blue) was used for nuclear counterstaining. (H) The subcellular distribution of Hnf4alpha was measured by nuclear-cytosolic fractionation of the hepatocytes and Western blotting. Histone H3 and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) are nuclear and cytoplasmic markers, respectively. All four lanes are from the

MA1-199

Antibody data