Fig. 2

An EGF-induced increase in the intracellular sodium level results in increased acetylated tubulin levels. a DAOY cells were incubated with 10 ng/ml EGF or vehicle for indicated time points. Equal amounts of protein were immunoblotted for total and activated (Y1173) EGFR, and acetylated α-tubulin. An immunoblot for total α-tubulin was included to ensure equal loading. b DAOY cells were incubated for one hour in Low Na⁺ buffer and then treated with 10 ng/ml EGF for indicated times. Immunoblots were performed for total and activated (Y1173) EGFR, total and activated Erk1/2, total and activated (S473) Akt and acetylated α-tubulin. The total α-tubulin immunoblot confirmed equal loading. c DAOY cells were incubated for one hour with 10 μM AG1478 prior to addition of 10 ng/ml EGF. Immunoblots were performed as in b. d DAOY cells were incubated in the absence of EGF with either AG1478 or PP2 for one hour before 50 μM ouabain or 100 nM gramicidin A were added for an additional three hours. Equal amounts of protein were immunoblotted for acetylated and total α-tubulin. e DAOY cells were pre-incubated with BAPTA-AM and then 10 ng/ml EGF was added for indicated times. Equal amounts of protein were immunoblotted for acetylated α-tubulin. An immunoblot for total α-tubulin ensured equal loading.