Fig. 3: Overexpression of constitutively active IKKα activates ERK signaling. | npj Precision Oncology

Fig. 3: Overexpression of constitutively active IKKα activates ERK signaling.

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

Fig. 3

a, b Human lung cancer cells, HCC827 (a) and H1650 (b), transfected with GFP (control), constitutively active IKKα, or kinase-dead IKKα were lysed using 1× RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts of proteins were separated with 4–20% SDS-PAGE and transferred to polyvinyl difluoride membranes. Antigen-coated membranes were incubated overnight with primary antibodies against IKKα, phosphorylated DARPP-32 (Thr-34), total DARPP-32, phosphorylated PP1α (Thr320), total PP1α, phosphorylated ERK (Thr202/Tyr204), total ERK, and α-tubulin (loading control). c, d Vehicle (DMSO)- or calyculin A-treated human HCC827 (c) and H1650 (d) cells were lysed with 1× RIPA buffer and subjected to immunoblotting using anti-phospho PP1α (Thr320), -total PP1α, -phospho ERK (Thr202/Tyr204), -total ERK, -DARPP-32, and -α-tubulin (loading control) antibodies. Chemiluminescence signals were detected after incubating membranes with HRP-tagged secondary antibodies. Representative images from one experiment are shown, but results were validated by performing three independent biological repeats. Bar graphs at the right show quantification of the results from the three western blotting experiments. Error bars indicate SEM. *P < 0.05; ns not significant.

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