Fig. 4.

Effect of endogenous PS2 on ER–mitochondria coupling. SH-SY5Y cells coexpressing mit-RFP and ER-GFP (or cyt/mit-Aeq) and treated with PS2-specific or control siRNA (20 nM) were analyzed by confocal microscopy (A and B) (Scale bar: 10 μm) to visualize the ER–mitochondria network, as described in Fig. 3 or by Western blotting (C) to check the expression level of PS2 (the PS2 C-terminal fragment, PS2-CTF, is shown). (D) The same cells were used to estimate the [Ca²⁺]_c and [Ca²⁺]_m responses (with aequorins) upon endogenous PS2 down-regulation with the protocol described in Fig. 1. Bars represent mean [Ca²⁺]_c (Left) and [Ca²⁺]_m (Right) peaks (mean ± SEM; ***P < 0.001; unpaired Student's t test; n = number of independent experiments).