Figure 3.

Knockdown of NAG causes the release of ZW10-RINT-1 from membranes as well as from p31. HeLa cells were mock-transfected or transfected with NAG (4160) or p31 siRNA. (A) At 72 h after transfection, cell lysates were prepared and immunoprecipitated with an antibody against syntaxin 18 (lanes 3 and 4) or p31 (lanes 5 and 6). The precipitated proteins were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (B) At 72 h after transfection, the cells were homogenized by passage 40 times through a 27-gauge needle. The homogenate was centrifuged at 1100 × g for 5 min to obtain the postnuclear supernatant (PNS; lanes 1–3) and then at 135,000 × g for 30 min to separate the membrane (lanes 4–6) and cytosol (lanes 7–9). (C) At 72 h after transfection, the cells were stained with the indicated antibodies. Note that many and some RINT-1-negative large puncta were observed in NAG- and p31-depleted cells, respectively (bottom row). Bars, 10 μm.