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Figure S5. Effects of VIPL knockdown on YE(gly)2 diffusion. (A) PNGaseF treatment of YE(gly)2. COS7 cells cultured in a 24-well plate were transfected with a YE(gly)2 expression vector using Fugene6. After 20 h, cells were lysed with 50 µl of 1% SDS and10 mM Tris-Cl, pH 7.5. The lysates were then diluted with 200 µl of 2% Triton X-100 and10 mM Tris-Cl, pH 7.5, and were treated with 0 (left) or 250 U (middle) PNGaseF (New England BioLabs, Inc.) for 15 min at 37°C. Total proteins were recovered and analyzed by Western blotting using anti-GFP antibody. The partially digested YE(gly)2 was compared with YE (right). Positions of singly and doubly glycosylated YE are indicated. Under these conditions, >80% of YE was doubly glycosylated. (B) Immunoblot of Y2E. As in A, either Y2E or YE was expressed, and the molecular weights were confirmed by Western blotting with an anti-GFP antibody. (C) Effect of VIPL siRNA on autocorrelation function of YE(gly)2. Control or VIPL siRNA was transfected into cells using Lipofectamine 2000, and the YE(gly)2 expression vector was bead loaded 70 h after transfection. FCS measurements were obtained as in Fig. 3. Mean autocorrelation curves from 59 (control siRNA) or 76 (VIPL siRNA) measurements are shown with error bars (95% confidence interval). The right panel shows an enlargement of a portion of the decay curve. We assumed that each autocorrelation function was composed of the three components identified in Fig. 3; best-fit percentiles to these components are shown in the table at the bottom. Knockdown efficiency was examined by estimating the amount of VIPL mRNA using glyceraldehyde-3-phosphate dehydrogenase mRNA as an internal control. Details are described in Materials and methods.