FIGURE 9.

Monomeric vGAAP protects cells from apoptosis and decreases the Ca²⁺ content of intracellular stores. U2-OS cells were transduced with a bicistronic lentivirus encoding GFP only, or GFP with vGAAP-HA, vGAAP-HA C9S, vGAAP-HA C9S/C60S, or Bcl-x_L-FLAG. A, after cell sorting to isolate GFP-expressing cells, lysates were immunoblotted (IB) with anti-GFP, anti-HA, and anti-FLAG antibodies. The sizes of molecular mass markers are shown in kDa. B, anti-HA antibody was used to detect GAAP and anti-GM130 antibody was used as a Golgi marker in cells prior to cell sorting. Cells were imaged using confocal microscopy. Scale bar, 10 μm. C–E, U2-OS cells stably expressing wild type or mutant vGAAPs were stimulated with staurosporine (0.5 μm, 6 h; C), doxorubicin (3 μm, 48 h; D), or TNFα (10 ng/ml) with cycloheximide (CHX) (20 μg/ml) for 16 h (E), and caspase activity was monitored. Results shown are representative of three independent experiments. F and G, typical increases in [Ca²⁺]_i evoked by addition of ionomycin (1 μm) to populations of transfected HeLa cells in Ca²⁺-free medium (F). Summary results show peak increases in [Ca²⁺]_i normalized to parallel measurements from mock-transfected cells. Results show means ± S.E. from five independent experiments. Asterisks (C, D, and G) indicate values that are significantly different from control: *, p < 0.05; **, p < 0.01; ***, p < 0.001.