Fig. 2: IKKα phosphorylates DARPP-32 at Thr-34. | npj Precision Oncology

Fig. 2: IKKα phosphorylates DARPP-32 at Thr-34.

From: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

Fig. 2

a Human A549 cell lines stably overexpressing FLAG-tagged human DARPP-32 isoforms (DARPP-32 and t-DARPP) were lysed and subjected to immunoprecipitation using anti-FLAG antibody–conjugated agarose beads. Immunoprecipitated lysates were used to perform nonradioactive in vitro kinase assays following incubation with commercially available active IKKα protein. At the end, the reaction mixtures were subjected to immunoblotting using antibodies against DARPP-32 phosphorylated on Thr-34 or Thr-75 and total DARPP-32 protein. b, c Human HCC827, PC9, and H1975 lung adenocarcinoma cell lines retrovirally transduced with either FLAG-tagged human DARPP-32 (b) or t-DARPP (c) cDNA plasmids were lysed, immunoprecipitated, incubated with active IKKα protein, and subjected to western blotting using anti-phospho (Thr-34 or Thr-75) DARPP-32 and anti-DARPP-32 antibodies. Data from one experimental replicate are shown. The experiments were repeated three times independently; each circle in a bar represents one experiment. Error bars indicate SEM. *P < 0.05; ns not significant.

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