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. Author manuscript; available in PMC: 2013 Dec 14.
Published in final edited form as: Mol Cell. 2012 Nov 1;48(5):771–784. doi: 10.1016/j.molcel.2012.09.028

Fig. 2. EGF Increases PKM2 Expression in a PKC- and NF-κB-dependent Manner.

Fig. 2

A–D, H, Immunoblotting analyses were performed with the indicated antibodies.

(A) U87/EGFR cells were pretreated with Bis-I (2 μM), Go6976 (2 μM), NF-κB activation inhibitor II (7 μM), an AKT inhibitor (10 μM), or TBB (50 μM), followed by EGF (100 ng/ml) stimulation for 12 h.

(B) 293T cells transiently transfected with pCep4 EGFR and vectors expressing IKKβ S177/181A were treated with or without EGF (100 ng/ml) for 12 h.

(C) U87/EGFR cells stably transfected with pGIPZ expressing a control or a RelA shRNA were treated with or without EGF (100 ng/ml) for 12 h.

(D) RelA+/+, RelA−/−, or RelA−/− fibroblasts with reconstituted RelA expression were treated with or without EGF (100 ng/ml) for 12 h.

(E) U87/EGFR cells treated with or without EGF (100 ng/ml) for 12 h. ChIP assay was performed with an anti-RelA antibody for immunoprecipitation, followed by PCR with PKM promoter–specific primers.

(F) An oligonucleotide containing the putative WT or mutated NF-κB binding sequence was labeled using [γ-32P] ATP and T4 polynucleotide kinase. Nuclear extracts of U87/EGFR cells treated with or without EGF (100 ng/ml) for 12 h were incubated with the 32P-labeled probe in the presence or absence of an anti-RelA antibody or unlabeled oligonucleotide. Samples were subjected to 5% polyacrylamide gel electrophoresis, and the dried gel was exposed to x-ray film.

(G) The luciferase reporter vector pGL3-promoter containing either the WT or a mutated PKM promoter fragment was transfected into U87/EGFR cells (left panel), or RelA+/+, RelA−/−, or RelA−/− fibroblasts with reconstituted RelA expression (right panel), which were treated with or without EGF (100 ng/ml) for 12 h. The relative levels of luciferase activity were normalized to the levels of untreated cells and to the levels of luciferase activity of the Renilla control plasmid. Data represent the mean ± standard deviation of three independent experiments.

(H) U251 cells infected with lentiviruses expressing a control or a PTBP1 shRNA were treated with or without EGF (100 ng/ml) for 12 h.

See also Figs. S1 and S2.