Figure 3
From: The PRKD1 promoter is a target of the KRas-NF-κB pathway in pancreatic cancer
Oncogenic Kras induces PRKD1 expression via the canonical NF-κB pathway.
(A) BxPC3 cells were transfected with vector control, KRasG12V or p65 as indicated. 24 hours after transfection lysates were analyzed by Western blotting for expression of endogenous PKD1 (anti-PKD1). Probing lysates for KRasG12D (anti-FLAG), p65 (anti-p65) or β-actin (anti-β-actin) served as expression or loading controls. (B) BxPC3 cells were transfected with vector control or p65 and PRKD1-luciferase and renilla-luciferase reporters. 24 hours after transfection cells were lysed and reporter gene assays performed. In addition, probing lysates for p65 (anti-p65) or β-actin (anti-β-actin) served as expression or loading controls. (C) BxPC3 cells were co-transfected with vector control, IκBα.SD or KRasG12V and PRKD1-luciferase and renilla-luciferase reporters. 24 hours after transfection cells were lysed and reporter gene assays performed. Probing lysates for IκBα (anti-IκBα), KRasG12V (anti-FLAG) or β-actin (anti-β-actin) served as expression or loading controls. (D) BxPC3 cells were co-transfected with vector control or KRasG12V and PRKD1-luciferase and renilla-luciferase reporters and, after 5 hours, treated with BMS345541 (10 μM). 24 hours after stimulation cells were lysed and reporter gene assays performed. Probing lysates for KRasG12V (anti-FLAG) or β-actin (anti-β-actin) served as expression or loading controls. (E) Panc1 cells were co-transfected with PRKD1-luciferase and renilla-luciferase reporters and then treated with BMS345541 (10 μM). 24 hours after stimulation cells were lysed and reporter gene assays performed. Probing lysates for β-actin (anti-β-actin) served as internal control. (F) Panc1 cells were transfected with control-shRNA or shRNA targeting KRas, as well as vector control or p65 and PRKD1-luciferase and renilla-luciferase reporters, as indicated. 24 hours after transfection cells were lysed and reporter gene assays performed. Probing lysates for KRas (anti-KRas), p65 (anti-p65) or β-actin (anti-β-actin) served as expression or loading controls.
