Anti-PDGFR alpha + Beta (phospho Y572 + Y574) antibody

• ICC

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Immunocytochemistry - Anti-PDGFR alpha + Beta (phospho Y572 + Y574) antibody (AB5443)

Immunocytochemistry analysis of 70% confluent log phase MCF7 cells treated with PDGF (50 ng/mL for 10 min) labeling PDGFR alpha + Beta (phospho Y572 + Y574) with ab5443. The 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. The cells were labeled with ab5443 at 2 μg/mL 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). Nuclei (Panel b : blue) were stained with DAPI. F-actin (Panel c : red) was stained with Alexa Fluor^® 594 Phalloidin. Panel d is a merged image showing cytoplasmic and membrane localization. Panel e shows untreated cells. Panel f shows no primary antibody control. The images were captured at 20X magnification.

• WB

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Western blot - Anti-PDGFR alpha + Beta (phospho Y572 + Y574) antibody (AB5443)

Peptide Competition : Extracts prepared from NIH3T3 cells left unstimulated (1) and stimulated with PDGF (2-5) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C, then were incubated with 0.50 μg/mL ab5443 antibody for two hours at room temperature in a 1% BSA-TBST buffer, following prior incubation with : no peptide (1, 2), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphotyrosine containing peptide (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar method. The data show that only the peptide corresponding to ab5443 blocks the antibody signal, thereby demonstrating the specificity of the antibody. Peptide Competition : Extracts prepared from NIH3T3 cells left unstimulated (1) and stimulated with PDGF

All lanes:

Western blot - Anti-PDGFR alpha + Beta (phospho Y572 + Y574) antibody (ab5443)

Predicted band size: 124 kDa

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