QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

Help viewing high resolution images Return to article

Fig. 7. Nocodazole treatment activates SOCE and I_CRAC in cells overexpressing EYFP-STIM1. (A) Cells overexpressing EYFP-STIM1 were treated with 10 µM nocodazole (NZL; red trace) or 0.1% DMSO (black trace) for 20 minutes in a nominally Ca²⁺-free extracellular solution, followed by addition of 1.8 mM extracellular Ca²⁺ in the absence of store depletion. Also shown is nocodazole-treated EYFP-STIM1-overexpressing cells in which Orai1 expression was reduced by RNAi (blue trace). Each trace represents the average response of all cells on a single coverslip (20-30 cells). (B) Cells expressing EYFP were treated with 10 µM nocodazole (red trace) or 0.1% DMSO (black trace), and Ca²⁺ entry was evaluated in the absence of store depletion, as described in (A). (C) The average difference between the peak 340/380 value following Ca²⁺ addition and the 340/380 value just before Ca²⁺ addition was calculated for cells expressing EYFP-STIM1 treated with 0.1% DMSO (n=85, three coverslips), 10 µM nocodazole (n=167, six coverslips) and in EYFP-STIM1-expressing, Orai1-knockdown cells treated with nocodazole (n=62, three coverslips) for experiments performed as described in (A). The data are represented as the mean±s.e.m.; P values are based on one-way ANOVA. (D-F) The responses of all the cells measured on a single coverslip are shown for the averaged traces shown in (A); (D) EYFP-STIM1 cells treated with DMSO; (E) EYFP-STIM1 cells treated with 10 µM nocodazole; (F) EYFP-STIM1-overexpressing, Orai1-knockdown cells treated with nocodazole. (G) Cells overexpressing EYFP-STIM1 were patched with an intracellular solution clamped at 0 mM Ca²⁺. Break-in was performed in 10 mM extracellular Ca²⁺, and the extracellular solution was switched to divalent-free at intervals of 1 minute, with intervals of 30 seconds of 10 mM Ca²⁺ in between. (H) The same protocol as in C was performed using a 0 mM Ca²⁺ intracellular solution on a nocodazole-treated cell overexpressing EYFP-STIM1.

Return to article