Figure 2

SEC62 silencing and calmodulin antagonists affect cellular calcium homeostasis similarly. A, HeLa cells were transfected with SEC62- or control siRNA, after 96 h loaded with FURA2-AM, and subjected to Ca²⁺ imaging. After 60 s in Ca²⁺-free buffer, cells were treated with ophiobolin A (100 μM), TFP (10 μM), or buffer for 10 min, and then thapsigargin was added. The graphs represent the mean cytosolic Ca²⁺ concentration of 182 (control siRNA), 325 (control siRNA + ophiobolin A), 198 (control siRNA + TFP) or 82 cells (SEC62 siRNA). B, HeLa-CES2 cells were loaded with Fluo5N-AM. After 60 s in Ca²⁺-free buffer, ophiobolin A, TFP, or buffer was added, samples were measured for 2 min, then thapsigargin and after 5 min ionomycin was added. C, HeLa cells were transfected with SEC61A1- or control siRNA, after 96h loaded with FURA2-AM, and after 60 s in Ca²⁺-free buffer treated with ophiobolin A, TFP or buffer. After 10 min Ca²⁺-solution was added (2.5 mM free Ca²⁺). D, HeLa cells were transfected with SEC62- , SEC61A1-, a combination of both siRNAs, or control siRNA, loaded with FURA2-AM, incubated in Ca²⁺-free buffer and subsequently treated with thapsigargin. The graphs represent the mean cytosolic Ca²⁺ concentration of 547 (control siRNA), 353 (SEC62 siRNA), 495 (SEC61A1 siRNA), or 395 cells (SEC62 + SEC61A1 siRNA). The insert shows the silencing efficiency determined by western blot (n=3). E, SPR spectroscopy was performed with Sec61α N-terminal peptide (measuring-cell) and TRAM N-terminal peptide (control-cell). Ca²⁺-containing buffer (control) or purified Sec62-C-His in buffer with or without Ca²⁺was passed over both cells. The insert shows a peptide spot binding assay. Peptides spanning the N-terminus of Sec61α were synthesized on a cellulose membrane and incubated with Sec62-C-His (1 μM) in binding buffer. Bound protein was detected using anti-His-POD coupled antibody and visualized with a luminescence-imaging system.