Anti-EPO-R (phospho Y454 + Y456) antibody [RP23040088]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-EPO-R (phospho Y454 + Y456) antibody [RP23040088] (AB308034)

For immunofluorescence analysis, 70% confluent log phase HeLa cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature for detection of EpoR (phospho T454 + T456) using ab308034 at 1/500 dilution in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Goat anti-Rabbit IgG Secondary Antibody at a dilution of 1/400 for 30 minutes at room temperature (Panel a : green)

F-actin (Panel b : red) was stained with Alexa Fluor® 594 Phalloidin

Nuclei (Panel c : blue) were stained with DAPI

Panel d is a merged image showing cytoplasmic and membrane localization

Panel e shows competition with the EpoR (phospho T454 + T456) peptide

The images were captured at 20X magnification

• WB

Supplier Data

Western blot - Anti-EPO-R (phospho Y454 + Y456) antibody [RP23040088] (AB308034)

Western blot analysis of EpoR (phospho T454 + T456) was performed by loading 30 µg of HeLa, K-562 cell lysates (lane 1, 2) using a 4-12% Bis-Tris gel, electrophoresis system, and pre-stained protein standard. Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. EpoR (phospho T454 + T456) was detected at ~28 kDa using ab308034 at 1 : 500 dilution in 2.5% skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with the phosphopeptide (10 µg/mL) (lane 3, 4). Goat anti-Rabbit IgG - HRP Secondary Antibody at 1 : 5000 dilution was used and chemiluminescent detection was performed (ECL).

All lanes:

Western blot - Anti-EPO-R (phospho Y454 + Y456) antibody [RP23040088] (ab308034) at 1/500 dilution

Lane 1:

HeLa cell lysate at 30 µg

Lane 2:

K-562 cell lysates at 30 µg

Lane 3:

Phosphopeptide treated HeLa cell lysates at 30 µg

Lane 4:

Phosphopeptide treated K-562 cell lysates at 30 µg

Secondary

All lanes:

Goat anti-Rabbit IgG - HRP Secondary Antibody at 1/5000 dilution

Predicted band size: 55 kDa

Observed band size: 28 kDa

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