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. 2001 Feb 13;98(5):2865–2870. doi: 10.1073/pnas.041608898

Figure 3.

Figure 3

Ryanodine restores E4032A activity in 1B5 myotubes. 1B5 myotubes expressing E4032A were initially examined for functional responses to RyR agonists. E4032A expression was determined by immunocytochemical analysis after methanol fixation. A and B show the same field of cells after immunostaining using 34C antibody to reveal 25 cells expressing E4032A. (A) Only 2 of these E4032A-expressing cells responded to 40 mM caffeine (arrows). (B) After addition of 500 μM ryanodine for 30 min, 12 additional E4032A-expressing cells responded to a subsequent application of 40 mM caffeine (arrowheads). (C) Changes in intracellular calcium for the cell indicated by the asterisk in B are shown. This cell did not respond to the initial application of 40 mM caffeine (clear bar) or to the application of 500 μM ryanodine for 30 min (gray bar). After washout of the ryanodine, this cell responded to two consecutive applications of 40 mM caffeine. (D) A wtRyR1-expressing 1B5 cell tested with the same experimental paradigm as C responded to 40 mM caffeine and 500 μM ryanodine. However, further responses to two consecutive applications of 40 mM caffeine were reduced. (Bar = 0.05 340/380 units vs. 125 sec.)