Figure 5.

NAADP Regulates EGF Signaling

(A) Representative western blot using antibodies to phosphotyrosine (pY) 1068 EGFR (top) or total EGFR (bottom) and homogenates from HeLa cells. Cells were treated with either DMSO (0.1%) or Ned-19 (100 μM) overnight and serum starved for 1 hr prior to EGF (100 ng/mL) stimulation for the indicated times.

(B) Summary data analyzing pY-EGFR levels (normalized to total EGFR) in lysates quantified as a percentage of DMSO control 10 min after EGF stimulation. Data are from three independent treatments (± SEM).

(C and D) Similar to (A) and (B) except western blots were performed with antibodies to pY 204 ERK1/2 or total ERK1/2 (C). Data are from three independent treatments (± SEM; D).

(E) Pseudo-colored images of the fluorescence ratio of HeLa cells loaded with the Ca²⁺ indicator, Fura-2, and stimulated with EGF (100 ng/mL) for the times indicated. Cells were treated with DMSO (0.1%) or Ned-19 (100 μM) overnight. Scale bar, 50 μm.

(F–H) Summary data quantifying the time course (F), area under the curve (AUC) (G, normalized to DMSO) and maximal change (Δ[Ca²⁺]) (H, normalized to DMSO) of cytosolic Ca²⁺ levels after stimulation with EGF for the indicated time. Data are from two to four independent treatments (± SEM) analyzing 309–828 cells.

See also Figure S4.