Anti-MEK1 + MEK2 (phospho S222) antibody

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MEK1 + MEK2 (phospho S222) antibody (AB4750)

Immunofluorescent analysis of mouse embryonic fibroblasts labelled for MEK1/2 (pS222) using ab4750, phosphospecific antibody. The cell is actively dividing. Blue represents chromosomes in anaphase of mitotic cell division. Green shows mitotic spindle expressing phosphorylated MEK1/2.

• WB

Supplier Data

Western blot - Anti-MEK1 + MEK2 (phospho S222) antibody (AB4750)

Western blot analysis was performed on whole cell extracts of A549 in different conditions, observed a 45 kDa band corresponding to Phospho-MEK1/MEK2 (Ser222) was observed in untreated A549 lysate, the signal increased upon EGF treatment, and decreased upon pretreatment with the EGFR antagonist, Afatinib. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel, XCell SureLock™ Electrophoresis System and Novex® Sharp Pre-Stained Protein Standard. Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate.

All lanes:

Western blot - Anti-MEK1 + MEK2 (phospho S222) antibody (ab4750)

Lane 1:

A549 whole cell extract lysate at 30 µg

Lane 2:

A549 whole cell lysate treated with EGF (200 ng/mL for 10 min) at 30 µg

Lane 3:

A549 whole cell lysate treated with Afatinib (0.5 uM for 16 h) followed by EGF (200 ng/mL for 10 min) at 30 µg

Secondary

All lanes:

Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate at 0.25 µg/mL

Predicted band size: 43 kDa

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• WB

Supplier Data

Western blot - Anti-MEK1 + MEK2 (phospho S222) antibody (AB4750)

Peptide Competition and Stimulation Extracts of NIH3T3 cells untreated (lane 1) or treated with 50 ng/mL PDGF for 15 minutes (2-5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 4% BSA-TBST buffer overnight at 4°C, then incubated with the MEK1&2 (pS222) antibody for two hours at room temperature in a 1% BSA-TBST buffer, following various prior incubation conditions. After washing, the membrane was incubated with goat F (ab)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Pierce SuperSignal method. The data show that only the phosphopeptide corresponding to MEK1&2 (pS222) block the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of MEK1&2 (pS222) phosphorylation by the addition of PDGF to this cell system.

All lanes:

Western blot - Anti-MEK1 + MEK2 (phospho S222) antibody (ab4750)

Lane 1:

Untreated NIH3T3 cells

Lane 2:

PDGF treated NIH3T3 cells, with no peptide incubation

Lane 3:

PDGF treated NIH3T3 cells, incubated with non-phosphopeptide immunogen

Lane 4:

PDGF treated NIH3T3 cells, incubated with generic phosphoserine peptide.

Lane 5:

PDGF treated NIH3T3 cells, incubated with phosphopeptide immunogen.

Secondary

All lanes:

A goat F (ab)2 anti-rabbit IgG alkaline phosphatase

Predicted band size: 43 kDa

false