Figure 3. Phosphorylation of IP³R3s expressed in 3KO cells.

(A) DT40 cells (lanes 1-2), 3KO cells (lanes 3-4), or IP₃R3-expressing 3KO cells (lanes 5-12) were exposed to 1 μM forskolin plus 200 μM IBMX for 10 min as indicated, lysates were prepared, and IP₃R3s phosphorylated at S⁹³⁴ were immunoprecipitated with anti-pS934. Total IP₃R3 in lysates prior to immunoprecipitation (upper panel) or phosphorylated IP₃R3 in immunoprecipitates (lower panel) were assessed in immunoblots with TL3. (B) 3KO-SSS cells were exposed to 0-100 μM forskolin in the absence (upper panel) or presence (lower panel) of 200 μM IBMX for 10 min, and IP₃R3 phosphorylation was assessed as in A. (C) IP₃R3^SSS-expressing HEK cells were exposed to 0.01-3.0 μM forskolin for 10 min and IP₃R3 phosphorylation was assessed using anti-pS934 in immunoblots of cell lysates. (D) Traces of typical Ca²⁺ responses to forskolin (added at t = 0.5 min) in 3KO cells (dashed line) and 3KO-SSS cells (solid lines). (E) 3KO-SSS cells were incubated with or without 1μM forskolin plus 200 μM IBMX for 10 min as indicated and levels of IP₃R3 in cell lysates prior to (pre) and following (post) immunoprecipitation of phosphorylated IP₃R3 with anti-pS934 were assessed in immunoblots with TL3. The lack of difference in signal intensity in control lysates pre and post immunoprecipitation (lanes 1 and 2) shows that IP₃R3^SSS is not significantly phosphorylated, whereas the difference in signal intensity in stimulated cells (lanes 3 and 4; a reduction of ∼50%) shows that at least 50% of IP₃R3^SSS is phosphorylated.