Figure 4

Confirmation of the specificity of ER phospho-antibodies. (A) Serine to alanine mutations at ERα phosphorylation sites inhibit reactivity of phospho-specific antibodies. COS-1 cells cultured in DMEM growth supplemented with 10% FBS were transiently transfected with 500 ng of either wt ERα or serine to alanine substituted ERα expression plasmids (S47A, S282A, S294A, or S559A). 18 hours post-transfection, cells were lysed and subjected to Western immunoblot analysis utilizing custom polyclonal antibodies directed toward the individual phosphorylated ERα residues (S47, S282, S294, or S559) or monoclonal ERα antibody as indicated. α-p-S282, α-p-S294, or α-p-S559 antibodies did not recognize S282A, S294A, or S559A, respectively, indicating phospho-antibody specificity. Mutation of S47 failed to eliminate immunoreactivity of αp-S47. (B) In vitro λ phosphatase treatment of ERα inhibits immunoreactivity of ERα phospho-antibodies. Baculovirus expressed ERα was subjected to dephosphorylation by λ phosphatase for 30 minutes at 30°C and analyzed by Western immunoblot with antibodies against p-S282, p-S294, p-S559, and total ERα. Dephosphorylation inhibited immunoreactivity of, α-p-S282, α-p-S294, and α-p-S559 without impacting immunoreactivity of total ERα antibody.