Figure 8.

Effect of reducing endogenous SMAP1 protein levels on clathrin-dependent endocytosis. (A) Immunoblot analysis of endogenous SMAP1 protein levels in HeLa cells transfected with the control RNA (lane 1) or with the siRNA against SMAP1 (lane 2). After incubating the cells with the RNAs, their lysates were subjected to immunoblot analysis using the anti-SMAP1 peptide antibody. The 62-kDa band indicated by the arrow represents the SMAP1 protein. The 62-kDa mass is much larger than the calculated 48-kDa mass of SMAP1, probably because SMAP1 is relatively basic and migrates slowly. (B) The subcellular distribution of β-COP and AP-1 in siRNA-treated HeLa cells. The cells were transfected with the siRNA against SMAP1 and incubated with (bottom panel) or without (top panel) 5 μg/ml brefeldin A for 2 min before fixation. The fixed cells were stained with anti-β-COP and anti-AP-1 antibodies. (C) Effect of siRNA treatment on transferrin internalization. HeLa cells were transfected with control RNA or siRNA and incubated with Alexa488-transferrin before fixation. DIC images are included to visualize the cells. Bar, 20 μm.