Peptide Competition Lysates from Jurkat cells either untreated (1, 6), treated with hydrogen peroxide (2-5), or the ionophore A23187 (7) were resolved on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 3% milk-TBST buffer for one hour at room temperature, then were incubated with the ETS1[pS251] antibody overnight at 4°C in a 1% milk-TBST buffer, following prior incubation with: no peptide (1, 2, 6, 7), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphoserine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal™ method. The data show that only the peptide corresponding to ETS1 [pS251] blocks the signal, verifying the specificity of the antibody. In addition, antibody blocking was not seen using phosphopeptides to the pS257, pS282, pS285, or pSpS309/312 sites (not shown).
Peptide Competition Lysates from Jurkat cells either untreated (1, 6), treated with hydrogen peroxide (2-5), or the ionophore A23187 (7) were resolved on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 3% milk-TBST buffer for one hour at room temperature, then were incubated with the ETS1[pS251] antibody overnight at 4°C in a 1% milk-TBST buffer, following prior incubation with: no peptide (1, 2, 6, 7), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphoserine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal™ method. The data show that only the peptide corresponding to ETS1 [pS251] blocks the signal, verifying the specificity of the antibody. In addition, antibody blocking was not seen using phosphopeptides to the pS257, pS282, pS285, or pSpS309/312 sites (not shown).
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