Fig. 6. Suppression of hVps34 expression does not prevent membrane association of the early endosome marker EEA1. (A) Control and hVps34 KD cells were seeded at 100,000 cells/dish in 35 mm dishes. 24 hours later, cells were examined by immunofluorescence microscopy using an anti-EEA1 antibody. Control cells treated with 0.1 µM or 1 µM wortmannin for 1 hour are also shown. (B) Cytosol (S) and particulate (P) fractions were prepared from control cells, hVps34 KD cells, or control cells treated for 1 hour with the indicated concentrations of wortmannin (0.1 µM or 1 µM), as described in the Materials and Methods. Immunoblot analysis was performed with an antibody against EEA1 and the relative amount of EEA1 recovered in the P fraction is expressed as a ratio to the amount in the S fraction. The results are means (±s.e.m.) of three determinations performed on separate cultures. The asterisk indicates that the decrease relative to the control and KD cells was significant at P< 0.05 (Student's t test). Essentially the same results were obtained when the amount of EEA1 recovered in the P fraction was normalized to the amount of a membrane marker (calreticulin) recovered in the same fraction (not shown).