R 30 min with nocodazole (working concentration 10 mM) or cytochalasin-B (10 mg/ml). Drugs were purchased from Sigma. Cells were transfected with following plasmids: Exo70-GFP or mCherry, Exo84-GFP or -mCherry cloned in buy 223488-57-1 pcDNA3.1 plasmid; Cav1-GFP or mRFP constructs were a kind gift of Dr A. Helenius (ETH, Zurich) [31]; Cavin-1-mRFP was a kind gift of Dr C. ?Lamaze (Institut Curie, Paris) [3] and tubby-GFP was a kind gift L. Shapiro (Columbia University, New York) [20]. Transfections with plasmid DNA (1 mg) were carried out with Fugene (Roche) according to the manufacturer’ instructions. Cells were analyzed 24 h after transfection. Cells were transfected with two independent siRNAs (CAL-120 Dharmacon) specific for EXOC7 (Exo70) at a concentration 100 nM using Oligofectamine (Invitrogen). Sequences of the forward strand were as follows: EXOC7-duplexC: 59-GAGAUGACAUGCUGGACGUUU-39, EXOC7-duplexD: 59-GGAAGGCCAUUGUGCGACAUU-39. Mock or siRNA-treated cells were analyzed 3 days after transfection.Western blot and antibodiessiRNA-treated cells were lysed in lysis buffer (50 mMTris-HCl [pH 7.4], 150 mM NaCl, 1 Triton X-100, 1 mM EDTA, and protease inhibitors Mini complete, Roche) and centrifuged at 16,000 g for 10 min at 4uC. Supernatants were separated by SDSPAGE and analyzed by immunoblotting. Rabbit polyclonal antibodies against actin (A5060) and Cav1 (610060) were purchased from Sigma and BD Bioscience, respectively. Monoclonal antibody against EXOC7 was a generous gift of Dr S. HsuImmunofluorescence analysisFor immunofluorescence analysis, Hela cells were plated on fibronectin coated coverslips, fixed and extracted with 0.3 Triton X-100 in 4 PFA for 20 min and further fixed for 20 min by 4 PFA. Then, cells were incubated with Cav1 antibodies in PBS and washed with PBS. Bound antibodies were detected with Cy3-conjugated mouse antibodies. Cells were then mounted in ProLong Gold antifade reagent (Invitrogen) containing DAPI. Images were taken using Eclipse 90i Upright Microscope with aCharacterization of Trafficking of Caveolin-CCD Camera CoolSNAP HQ2 and a Piezo Flexure Objective Scanner. The system was steered by Metamorph 7 software.Supporting InformationFigure S1 TIRF-M revealed specific structures positive for Cav1-GFP and Exo70-mCherry. (A) Hela cells expressing Cav1-GFP and Exo70-mCherry were visualized by TIRF-M (top panel). The bottom panel shows a wide-field image of the same field. B) Hela cells expressing tubby-GFP and Exo70-mCherry were visualized by TIRF-M. Scale bars, 5 mm. Inset shows higher magnification of region indicated by an arrow. (EPS) Figure S2 Caveolin1 co-localizes with late endosomes markers, GFP-rab7 or GFP-VAMP7. (A) Hela cells expressing Cav1-mRFP and GFP-rab7 were detached and maintained in suspension for 1 h in culture medium at 37uC, and then replated on fibronectin for 3 h and visualized using time-lapse confocal spinning disk microscopy (upper panel). The lower panels represent selected frames from the time-lapse series (time is given in second). Arrows point to a Cav1-, rab7-positive intracellular vesicle. 11967625 (B) Hela cells expressing Cav1-mRFP and GFP-VAMP7 were treated and analyzed as in panel A. Scale bars, 5 mm. (EPS) Figure S3 Exo70 silencing. Samples of Hela cells treated with two independent Exo70 siRNAs (7c and 7d) were analyzed by western blotting with the indicated antibodies. Levels of Exo70 were reduced by 1.9 and 2.2-fold upon treatment with Exo70 siRNA 7c and 7d, respectively. Actin immunoblotting stai.R 30 min with nocodazole (working concentration 10 mM) or cytochalasin-B (10 mg/ml). Drugs were purchased from Sigma. Cells were transfected with following plasmids: Exo70-GFP or mCherry, Exo84-GFP or -mCherry cloned in pcDNA3.1 plasmid; Cav1-GFP or mRFP constructs were a kind gift of Dr A. Helenius (ETH, Zurich) [31]; Cavin-1-mRFP was a kind gift of Dr C. ?Lamaze (Institut Curie, Paris) [3] and tubby-GFP was a kind gift L. Shapiro (Columbia University, New York) [20]. Transfections with plasmid DNA (1 mg) were carried out with Fugene (Roche) according to the manufacturer’ instructions. Cells were analyzed 24 h after transfection. Cells were transfected with two independent siRNAs (Dharmacon) specific for EXOC7 (Exo70) at a concentration 100 nM using Oligofectamine (Invitrogen). Sequences of the forward strand were as follows: EXOC7-duplexC: 59-GAGAUGACAUGCUGGACGUUU-39, EXOC7-duplexD: 59-GGAAGGCCAUUGUGCGACAUU-39. Mock or siRNA-treated cells were analyzed 3 days after transfection.Western blot and antibodiessiRNA-treated cells were lysed in lysis buffer (50 mMTris-HCl [pH 7.4], 150 mM NaCl, 1 Triton X-100, 1 mM EDTA, and protease inhibitors Mini complete, Roche) and centrifuged at 16,000 g for 10 min at 4uC. Supernatants were separated by SDSPAGE and analyzed by immunoblotting. Rabbit polyclonal antibodies against actin (A5060) and Cav1 (610060) were purchased from Sigma and BD Bioscience, respectively. Monoclonal antibody against EXOC7 was a generous gift of Dr S. HsuImmunofluorescence analysisFor immunofluorescence analysis, Hela cells were plated on fibronectin coated coverslips, fixed and extracted with 0.3 Triton X-100 in 4 PFA for 20 min and further fixed for 20 min by 4 PFA. Then, cells were incubated with Cav1 antibodies in PBS and washed with PBS. Bound antibodies were detected with Cy3-conjugated mouse antibodies. Cells were then mounted in ProLong Gold antifade reagent (Invitrogen) containing DAPI. Images were taken using Eclipse 90i Upright Microscope with aCharacterization of Trafficking of Caveolin-CCD Camera CoolSNAP HQ2 and a Piezo Flexure Objective Scanner. The system was steered by Metamorph 7 software.Supporting InformationFigure S1 TIRF-M revealed specific structures positive for Cav1-GFP and Exo70-mCherry. (A) Hela cells expressing Cav1-GFP and Exo70-mCherry were visualized by TIRF-M (top panel). The bottom panel shows a wide-field image of the same field. B) Hela cells expressing tubby-GFP and Exo70-mCherry were visualized by TIRF-M. Scale bars, 5 mm. Inset shows higher magnification of region indicated by an arrow. (EPS) Figure S2 Caveolin1 co-localizes with late endosomes markers, GFP-rab7 or GFP-VAMP7. (A) Hela cells expressing Cav1-mRFP and GFP-rab7 were detached and maintained in suspension for 1 h in culture medium at 37uC, and then replated on fibronectin for 3 h and visualized using time-lapse confocal spinning disk microscopy (upper panel). The lower panels represent selected frames from the time-lapse series (time is given in second). Arrows point to a Cav1-, rab7-positive intracellular vesicle. 11967625 (B) Hela cells expressing Cav1-mRFP and GFP-VAMP7 were treated and analyzed as in panel A. Scale bars, 5 mm. (EPS) Figure S3 Exo70 silencing. Samples of Hela cells treated with two independent Exo70 siRNAs (7c and 7d) were analyzed by western blotting with the indicated antibodies. Levels of Exo70 were reduced by 1.9 and 2.2-fold upon treatment with Exo70 siRNA 7c and 7d, respectively. Actin immunoblotting stai.
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