FIGURE 1.

H- and K-Ras anchors target annexin A6 to the PM independent of Ca²⁺. A, annexin A6YFP-tH, annexin A6YFP-tK, GFP-tK, and annexin A1YFP were expressed in HEK293 cells. 48 h post-transfection the cells were collected, lysed, and analyzed by SDS-PAGE followed by Western blotting with monoclonal antibodies against GFP or human annexin A6. Lanes 1 and 5 show A6YFP-tH, lanes 2 and 6 A6YFP-tK, lanes 3 and 7 GFP-tK, and lanes 4 and 8 annexin A1YFP. The positions of endogenous annexin A6 (wt A6) and A6-fusions are indicated by arrows. B, live HEK 293 cells expressing annexin A6GFP-tK were examined at the confocal microscope under resting conditions (low basal [Ca²⁺]_i). Cells are shown in orthogonal projection, bar = 5 μm. C, membranes from HEK293 cells expressing annexin A6YFP-tH, or annexin A6YFP-tK, were isolated in the presence of 0.2 mm Ca²⁺. The purified membranes were resuspended in Na⁺-Tyrode buffer containing 0.2 mm Ca²⁺, or 1 mm EGTA, as indicated. Membrane-bound annexins were separated from the unbound proteins by centrifugation (20 min, 12,000 rpm at 4 °C). The membrane-bound proteins in the pellet (P) and the soluble proteins in the supernatant (S) were analyzed by SDS-PAGE followed by Western blotting with monoclonal antibodies against annexin A6. Position of endogenous annexin A6 and A6YFP-tH and -tK fusions is indicated by arrows.