Figure 5.

Effect of cell permeabilization on release of resident organellar proteins. Vero cells were either homogenized or permeabilized with SLO as described in Materials and Methods. For homogenization, the cells were removed from the plastic by scraping and disrupted in PBS by passing them 10 times up and down through a 0.5-mm gauge syringe. The suspension was then centrifuged for 10 min in an Eppendorf centrifuge to remove nuclei and mitochondria. The postmitochondrial supernatant as well as the buffer fraction obtained after the SLO treatment were centrifuged at 128,000 × g for 30 min. In both cases, the pellet (P) was taken as the membrane fraction and the supernatant (B, buffer) as the cytosolic fraction. Both fractions were fractionated by SDS/PAGE, transferred to Immobilon membranes, and probed with antibodies against GRP94, BIP, HSP70, PDI, p58, and rab5.