Supplemental Data Caveolin-Stabilized Membrane Domains as Multifunctional Transport and Sorting Devices in Endocytic Membrane Traffi c

(A) HeLa cells transiently expressing GFP-tagged Rabs were incubated for 2 hr with Alexa Fluor-labeled SV40 (AF-SV40), fi xed, and analyzed with confocal microscopy. The percentage of AF-SV40 signal overlapping with the GFP-Rab signal was calculated as published (Pelkmans et al., 2001). As a control, overlap with Cav-1-GFP in a HeLa cell line stably expressing low levels was determined. SV40 did not signifi cantly overlap with most GFP-Rabs, with the exception of GFP-Rab5a, but some was observed to overlap, most signifi cantly with Rab5a. Scale bars, 20 μm. (B) HeLa cells transiently transfected with a mixture of GFP-rab5Q79L and RFP-Eps15EΔ95/295 were allowed to internalize 200 ng Alexa Fluor 647-labeled Transferrin (AF647-Tfn) for 10 min. A fi eld is depicted that contained a GFPrab5Q79L-transfected cell, a GFPrab5Q79L- and RFP-Eps15EΔ95/295-transfected cell, and a RFP-Eps15EΔ95/295-transfected cell, surrounded by nontransfected cells. While Tfn internalization is stimulated in the GFPrab5Q79L-transfected cell, it is reduced in the GFPrab5Q79L- and RFP-Eps15EΔ95/295-transfected cell. Quantifi cation of the Tfn signal in fi ve to 18 cells shows that Eps15EΔ95/295 can still block Tfn endocytosis by 65%–70 % in the presence of rab5aQ79L. Most potent block in Tfn uptake was found in the Eps15EΔ95/295-transfected cell. Scale bar, 20 μm. (C) (Left) Western blot detection of caveolin-1 in whole-cell lysates of HeLa cells and the stably expressing cav1-GFP cell line derived from them. The level of cav1-GFP is slightly less then that of endogenous caveolin-1. (Middle) Schematic of procedure to obtain optical sections of cells used for quantifi cation of overlap between molecules and to represent morphological information by maximum projection or surface rendering. (Right) HeLa cells stably expressing Cav1-GFP were fi xed and immunostained against EEA1 and Giantin to label early endosomes and the Golgi complex, respectively. The maximum intensity of a Z stack of 40 deconvolved sections is depicted. In the image, all signal depicted come