Anti-Amphiregulin antibody [EPR28751-564]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Amphiregulin antibody [EPR28751-564] (AB323476)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized AREG KO HCT116 (human colorectal carcinoma epithelial cell) cells labelling Amphiregulin with ab323476 at 1/100 (5.14 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2ug/ml) dilution (Green).

Confocal image showing cytoplasmic staining in HCT 116 cell line treated with PMA (100 ng/ml) for 48 hours, then add BFA (300 ng/ml) for 24 hours (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2ug/ml) dilution.

• WB

Supplier Data

Western blot - Anti-Amphiregulin antibody [EPR28751-564] (AB323476)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

20 kDa mature cleaved form of amphiregulin and 40 kDa precursor form of amphiregulin ADAM17 are observed.

The expression profile is consistent with that described in the literature (PMID : 21658434).

The expression of Amphiregulin isupregulated in response to PMA/TPA treatment (PMID : 2325643).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Amphiregulin antibody [EPR28751-564] (ab323476) at 1/1000 dilution

Lane 1:

Untreated MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

MCF7 treated with 100ng/ml PMA for 48 hours, 300ng/ml BFA was then added for additional 24 hours whole cell lysate at 50 µg

Lane 3:

Untreated HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 50 µg

Lane 4:

HepG2 treated with 100ng/ml PMA for 48 hours, 300ng/ml BFA was then added for additional 24 hours whole cell lysate at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 20 kDa,40 kDa,36 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-Amphiregulin antibody [EPR28751-564] (AB323476)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

20 kDa mature cleaved form of amphiregulin and 40 kDa precursor form of amphiregulin ADAM17 are observed.

The expression profile is consistent with that described in the literature (PMID : 21658434).

Performed under reducing conditions.

In Western blot, ab323476 was shown to bind specifically to AREG . Target of interest was observed at 20 kDa and 40 KDa in wild-type HCT 116 cell lysates (lane 2) with no signal observed at this size in AREG knockout cell line (lanes 3-4) (lane 3, knockout cell line ab289175).

The expression of Amphiregulin is upregulated in response to PMA/TPA treatment (PMID : 2325643).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Amphiregulin antibody [EPR28751-564] (ab323476) at 1/1000 dilution

Lane 1:

Untreated Wild-type HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Wild-type HCT 116 treated with 100ng/ml PMA for 48 hours, 300ng/ml BFA was then added for additional 24 hours whole cell lysate at 20 µg

Lane 3:

Untreated AREG knockout HCT 116 whole cell lysate at 20 µg

Lane 4:

AREG knockout HCT 116 treated with 100ng/ml PMA for 48 hours, 300ng/ml BFA was then added for additional 24 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 20 kDa,40 kDa,36 kDa

false

Exposure time: 26s