Supplementary Material & Methods
Preparation of enriched plasma membrane fractions:
Cells were harvested and washed twice with PBS. Pellets (5x106 cells/pellet) were frozen at -80ºC until
further use.
Upon thawing cells were resuspended in 500 μl of lysis buffer [50 mM Tris (pH 7.8); 250 mM Sucrose; 2
mM EDTA] with protease inhibitors and incubated on ice for 10 min. Cells were lysed with 30 passes
through the 301/2 Gauge needle at 4°C.
The cell debris, unbroken nuclei, and other membrane proteins were removed by centrifugation at
1,000 g for 10 min at 4ºC (pellet 1). The supernatant was layered onto sucrose buffer 1:1 [60% sucrose,
10 mM Tris-HCl (pH7.4)] and centrifuged at 160,000 g for 70 min at 4ºC (34460 rpm, rotor SW60Ti,
centrifuge Beckman Optima LE-80K).
The supernatant (SP1) was discarded and the membrane fraction (interface) on top of the sucrose
cushion (SC) was collected and diluted 1:2 with 50 mM Tris (pH 7.8) and centrifuged at 100,000 g for 1 h
at 4ºC (40,000 rpm, rotor TLA55, centrifuge Beckman Optima TLX). Again, the supernatant (SP2) was
discarded and the pellet washed with 100 mM Na2CO3 (pH11.5) for 2h at 4°C followed by
ultracentrifugation at 100,000 g for 90 min at 4°C.
Finally, the resultant supernatant (SP3) was discarded and the pellet (plasma membrane enriched
fraction; PM) was rinsed twice with cold water at 20,000 g for 30 min at 4°C (rotor TLA55, centrifuge
Beckman Optima TLX).
For protein quantification pellets were dissolved in 5% SDS, 62,5 mM Tris-HCl pH6.8 and protein content
of the membrane fraction measured using BCA™ Protein Assay Kit (Pierce).
Western-blot experiments:
After quantification each fraction (25 µg/well and 50 ug for total protein were loaded) was run in a 412% NuPAGE® Novex® Bis-Tris gel (1.5 mm, 15 well), using NuPAGE® MOPS SDS Running Buffer (Life
Technologies). Proteins were stained with Instant Blue (Expedeon). Gel transfer was performed in a
semi-dry transfer unit (Hoeffer TE-70, Amersham Biosciences) at 1.5 mA /cm2 for 80 minutes. Gels were
transferred to PVDF (Millipore) membranes and blocked ON with 5 % (w/v) milk in 0.01 % (v/v) Tween,
Tris-buffered saline solution. The following primary antibodies were used: goat anti-HCAM (1:200
dilution) from Santa Cruz Biotechnology, rabbit anti-CD71 (1:250) from Santa Cruz Biotechnology, mouse
anti-GS28 (1:100) from BD Transduction Laboratories, mouse anti-ERGIC53 (1:250) from Enzo Life
Sciences, rabbit anti-Calnexin (1:8000) from Santa Cruz Biotechnology, rabbit anti-COX11 (1:1000) from
Santa Cruz Biotechnology, mouse anti-GM130 (1:200) from BD Transduction Laboratories and mouse
anti-Alfa-Tubulin (1:5000) from Sigma. Secondary antibodies (HRP conjugated) used were: rabbit antigoat (1:5000), donkey anti-mouse (1:5000) and donkey anti-rabbit (1:5000), all from GE Healthcare.
Detection was performed with Amersham ECL Plus (GE Healthcare) and images were acquired with
ChemiDoc.