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. 2013 May 21;8(5):e63729. doi: 10.1371/journal.pone.0063729

Figure 4. PP2A-B56γ1 binds to and dephosphorylates pTpYERK.

Figure 4

(A) Pull down assay. HA-tagged B56 protein in B56-transfected Calu1 cells was immunoprecipitated with anti-HA antibodies. pTpYERK, IER3 and B56s in the immune complex were detected by western blotting. Similarly, the complex immunoprecipitated from 3T3-B56γ1 cells with rabbit anti-pTpYERK antibodies was run in polyacrylamide gel under non-reducing conditions and B56γ1 was detected by western blotting using rabbit anti-mouse B56γ1 antibodies and HRP-labeled anti-rabbit IgG antibodies. (B) Immune complex phosphatase assay. Immobilized PP2A trimer co-immunoprecipitated with anti-HA antibodies on protein G-Sepharose beads was used as an enzyme and pTpYERK-rich cell lysate of Calu1 cells stimulated with EGF was used as a substrate. Beads suspensions were incubated for 30 min in the crude substrate solution. pTpYERK in the reacted lysate was detected by western blotting. Immunoprecipitated enzyme was quantitated by western blotting using rat anti-HA antibodies.