Anti-NFATC4 antibody [EPR25678-90]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-NFATC4 antibody [EPR25678-90] (AB324108)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HaCaT (human skin keratinocyte, Left) / HeLa (human cervical adenocarcinoma epithelial cell, Right) cells labelling NFATC4 with ab324108 at 1/500 dilution (0.1 ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Low expression : HaCaT (PMID : 27893713).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-NFATC4 antibody [EPR25678-90] (AB324108)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HepG2 (human hepatocellular carcinoma epithelial cell, Left) / A-204 (human muscle rhabdomyosarcoma cell, Right) cells labelling NFATC4 with ab324108 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Low expression : HepG2.

• IP

Supplier Data

Immunoprecipitation - Anti-NFATC4 antibody [EPR25678-90] (AB324108)

NFATC4 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab324108 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab324108 at 1/1000 dilution.

Blocking and dilution buffer and concentration : 5% NFDM/TBST

To minimize protein degradation, cells were lysed immediately after harvest and then applied for Immunoprecipitation as soon as possible.

All lanes:

Immunoprecipitation - Anti-NFATC4 antibody [EPR25678-90] (ab324108) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

ab324108 at 1/30 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab324108 in HUVEC whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Observed band size: 80-150 kDa

false

Exposure time: 67s

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-NFATC4 antibody [EPR25678-90] (AB324108)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling NFATC4 with ab324108 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• WB

Supplier Data

Western blot - Anti-NFATC4 antibody [EPR25678-90] (AB324108)

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

The expression profile/molecular weight observed is consistent with what has been described in the literature (PMID : 35847954; PMID : 17577925; PMID : 34445390).

Low expression : HepG2, HaCaT (PMID : 27893713).

To minimize protein degradation, cells/tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

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

All lanes:

Western blot - Anti-NFATC4 antibody [EPR25678-90] (ab324108) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (Frozen lysate) at 20 µg

Lane 2:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (Fresh lysate) at 20 µg

Lane 3:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate (Frozen lysate) at 20 µg

Lane 4:

HaCaT (human skin keratinocyte) whole cell lysate (Frozen lysate) at 20 µg

Lane 5:

Untreated A-204 (human muscle rhabdomyosarcoma cell) whole cell lysate (Frozen lysate) at 20 µg

Lane 6:

A-204 treated with 10µM MG-132 for 4 hours whole cell lysate (Frozen 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: 80-150 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-NFATC4 antibody [EPR25678-90] (AB324108)

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

To minimize protein degradation, cells/tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

All lanes:

Western blot - Anti-NFATC4 antibody [EPR25678-90] (ab324108) at 1/1000 dilution

All lanes:

NIH/3T3 (mouse embryonic fibroblast) 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: 80-150 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-NFATC4 antibody [EPR25678-90] (AB324108)

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

This antibody does not cross-react with recombinant human NFATC1, NFATC2 or NFATC3 by western blot.

In Western blot, Anti-DDDDK-tag antibody - staining at 1/5000 dilution.

All lanes:

Western blot - Anti-NFATC4 antibody [EPR25678-90] (ab324108) at 1/1000 dilution

Lane 1:

DDDDK-tagged human NFATC1 recombinant protein at 10 ng

Lane 2:

DDDDK-tagged human NFATC2 recombinant protein  at 10 ng

Lane 3:

DDDDK-tagged human NFATC3 recombinant protein  at 10 ng

Lane 4:

Human NFATC4 fragment at 10 ng

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: 80-150 kDa,78 kDa,100 kDa,115 kDa

false

Exposure time: 180s