Anti-HNF-4-alpha antibody [EPR3648]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-HNF-4-alpha antibody [EPR3648] (AB92378)

Overlay histogram showing HepG2 cells stained with unpurifiedab92378 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (unpurified ab92378, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-HNF-4-alpha antibody [EPR3648] (AB92378)

Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling HNF-4 with purified ab92378 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/500) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1 : primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-HNF-4-alpha antibody [EPR3648] (AB92378)

Intracellular Flow Cytometry analysis of HepG2 cells labelling HNF-4 with purified ab92378 at 1/70 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-HNF-4-alpha antibody [EPR3648] (AB92378)

Immunocytochemistry/Immunfluorescence analysis of HepG2 cells labelling HNF-4-alpha with unpurified ab92378 at a 1/100 dilution.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-HNF-4-alpha antibody [EPR3648] (AB92378)

Chromatin was prepared from HepG2 (Human liver hepatocellular carcinoma cell line) cells. ChIP was performed with 10^7 HepG2 cells and 8 µg of ab92378 [EPR3648]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.

• WB

Lab

Western blot - Anti-HNF-4-alpha antibody [EPR3648] (AB92378)

Western blot : Anti-HNF4A antibody [EPR3648] (ab92378) staining at 1/2000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab92378 was shown to bind specifically to HNF4A. A band was observed at 50-55 kDa in wild-type A549 cell lysates with no signal observed at this size in HNF4A knockout cell line. To generate this image, wild-type and HNF4A knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-HNF-4-alpha antibody [EPR3648] (ab92378) at 1/2000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

HNF4A knockout A549 cell lysate at 20 µg

Lane 3:

HepG2 cell lysate at 20 µg

Lane 4:

HEK-293 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 50 kDa,55 kDa

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• WB

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Western blot - Anti-HNF-4-alpha antibody [EPR3648] (AB92378)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HNF-4-alpha antibody [EPR3648] (ab92378) at 1/5000 dilution

All lanes:

HepG2 cell lysate at 10 µg

Secondary

All lanes:

Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 53 kDa

Observed band size: 53 kDa

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• WB

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Western blot - Anti-HNF-4-alpha antibody [EPR3648] (AB92378)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HNF-4-alpha antibody [EPR3648] (ab92378) at 1/2000 dilution

All lanes:

SW480 cell lysate at 20 µg

Secondary

All lanes:

Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 53 kDa

Observed band size: 53 kDa

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• WB

Unknown

Western blot - Anti-HNF-4-alpha antibody [EPR3648] (AB92378)

All lanes:

Western blot - Anti-HNF-4-alpha antibody [EPR3648] (ab92378) at 1/1000 dilution

Lane 1:

HepG2 cell lysate at 10 µg

Lane 2:

A549 cell lysate at 10 µg

Lane 3:

SW480 cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 53 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-HNF-4-alpha antibody [EPR3648] (AB92378)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HepG2 (Human liver hepatocellular carcinoma cell line) cells and 5 µg of ab92378 [EPR3648]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. The ChIP data was conducted on chromatin prepared from HepG2 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 HepG2 cells and 8 µg of ab92378. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. Additional screenshots of mapped reads can be downloaded here. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

CiteAb

Western blot - Anti-HNF-4-alpha antibody [EPR3648] (AB92378)

HNF-4-alpha western blot using anti-HNF-4-alpha antibody [EPR3648] ab92378. Publication image and figure legend from Li, M., Wang, Y., et al., 2019, Cell Biosci, PubMed 31417670.

ab92378 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 ab92378 please see the product overview.

SRC-3 inhibits HNF4α-induced HBV core-promoter activity. a Ectopic expression of SRC-3 or CA-Akt inhibited HNF4α-induced HBV core-promoter activity in HepG2 cells. b Transfection of CA-Akt into SRC-3-knockdown cells inhibited HBV core promoter activity. c Transfection of CA-Akt into SRC-3-knockdown cells prevented the nuclear translocation of HNF4α. Control constructs were transfected into control cell or SRC-3-knockdown cells and CA-Akt were transfected into SRC-3-knockdown cells for 48 h, then cell fractionation was carried out and fractions were analyzed by western blot. All experiments were repeated at least 3 times independently. *p < 0.05, **p < 0.01

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