Fig. 1

Sodium influx induces an accumulation of acetylated tubulin. a DAOY cells were incubated with 50 μM ouabain or ethanol (vehicle) for indicated times. Equal amounts of protein were separated by SDS-PAGE and immunoblotted with antibodies recognizing acetylated lysines (upper panel) or acetylated α-tubulin (lower panel). b Immunoblot for acetylated α-tubulin of DAOY cells treated for three hours at indicated concentrations with the sodium ionophore gramicidin A or the Na,K-ATPase inhibitor ouabain. An immunoblot for total α-tubulin ensured equal loading. c DAOY cells were treated for three hours at indicated concentrations with the potassium ionophore valinomycin or the sodium ionophore monensin. Gramicidin A was included for comparison. Equal amounts of protein were used for immunoblotting using antibodies against acetylated α-tubulin. An immunoblot for total α-tubulin ensured equal loading. d DAOY cells were treated with ouabain or gramicidin A as indicated in isotonic, sodium containing buffer (High Na⁺) or in a low-sodium buffer in which sodium was substituted with rubidium (Low Na⁺). Cells were lysed one or three hours after treatment and equal amounts of protein were immunoblotted for total and acetylated α-tubulin. e DAOY cells were incubated for three hours with 50 μM ouabain or vehicle in High Na⁺ or in Low Na⁺ buffer. The cells were fixed and immunostained with acetylated α-tubulin and pan-α-tubulin antibodies. For easier comparison, parameters for image acquisition were kept constant between samples. Scale bar, 10 μm. f DAOY cells were pre-incubated with the intracellular calcium chelator BAPTA-AM and then ouabain or gramicidin A were added for three hours. Equal amounts of protein were immunoblotted for acetylated α-tubulin. An immunoblot for total α-tubulin ensured equal loading.