PKCα and PKCδ Regulate ADAM17-Mediated Ectodomain Shedding of Heparin Binding-EGF through Separate Pathways
Figure 4
PMA-induced HB-EGF shedding involves cooperative functions of PKCα and PKCδ.
A) Western blot analysis of PKCα, PKCδ, and PKCε in total cell extracts from AP-HB-EGF expressing HT1080 cells reverse transfected with control (C) siRNAs or siRNAs against the indicated PKC isoforms. α-Tubulin is used as an internal loading control. B) AP-HB-EGF release measured as alkaline phosphatase activity (OD 405 nm) in conditioned media and C) surface AP-HB-EGF measured as cell surface fluorescence intensity per cell (FLU/cell) in DMSO control-treated (C; black bars) and 30 min PMA-treated (PMA; white bars) in AP-HB-EGF expressing HT1080 cells 72 h after reverse transfection with control (C) siRNAs or siRNAs against the indicated PKC isoforms. D) Fold PMA-induced change in alkaline phosphatase activity (% of control) in conditioned media from AP-HB-EGF expressing HT1080 cells treated for 15 or 30 min with either low (25 nM) or high (400 nM) concentrations of PMA, 72 h after reverse transfection with the indicated siRNAs. All graphs show average values ± standard error of the mean of at least three independent experiments each done in triplicate. *p<0.05; **p<0.01, ***p<0.001 after one-way analysis of variance with Bonferroni's post tests for multiple comparisons. Unless otherwise indicated, the comparison is relative to the respective control.