The Rate of NF-κB Nuclear Translocation Is Regulated by PKA and A Kinase Interacting Protein 1
Figure 6
Blocking access of PKAc to p65 accelerates the kinetics of nuclear translocation of p65 in the presence of TNFα and augments p65 transcriptional activity.
HeLa cells transfected with A) GFP-p65 WT alone, or with B) CAT 1-29 peptide, C) mCherry AKIP 1A, or D) both CAT 1-29 peptide and mCherry AKIP 1A. Cells were stimulated with TNFα (1 ng/ml) for 0, 15, 30 or 45 min, then fixed and permeabilized. Data were collected as described in Methods and Materials. DAPI staining of the nucleus and merged images are shown for each of the conditions in lower panels. E) HEK 293 cells transfected with either mCherry AKIP 1A, scrambled CAT 1-29 peptide, CAT 1-29 peptide, or mCherry AKIP 1A and CAT 1-29 peptide were stimulated with TNFα for 0, 15, 30, or 45 min. Representative Western blots demonstrate the accumulation of endogenous p65 in the nucleus compared to total p65 levels. F) Quantification of nuclear p65 levels. The amount of nuclear p65 was calculated based on normalized values of total p65 protein. G) HeLa cells were transfected with a NF-κB luciferase reporter in the presence of either mCherry alone or mCherry AKIP 1A and stimulated for up to 1 h with TNFα (1 ng/ml). Increased basal and TNFα stimulated p65 transcriptional activity was observed in the presence of AKIP 1A.