Fig. 8. Knockdown of MLCK protein expression by siRNA does not inhibit rearrangement of EYFP-Stim1. (A) HEK293 cells left untransfected (WT; lane 1), or transfected with control siRNA (lane 2) or one of three different MLCK siRNA constructs (lanes 3-5) were subjected to western blotting with an antibody against MLCK. (B) Three independent experiments were performed as described in panel A, and the average band intensities for each condition are expressed as a percentage of wild-type±s.e.m.; ^*, significant difference compared with wild-type cells (P<0.01) based on one-way ANOVA. (C) Cells were transfected with control siRNA or (D) with MLCK #3, and TIRFM experiments were performed in which stores were depleted with 2 µM thapsigargin in nominally Ca²⁺-free extracellular solution. Each trace represents a single cell, and the responses of all the cells measured from a single coverslip are shown.