Rabbit anti caveolin 1

Western blots were performed using 1:10,000 mouse anti-β-catenin (BD Biosciences, 610153), 1:1000 mouse anti-vinculin (Sigma, SAB4200729), or 1:1000 rat anti-N-cadherin [Developmental Studies Hybridoma Bank, MNCD2, (Matsunami and Takeichi, 1995 (link))], 1:1000 rat anti-E-cadherin (Thermo, 14-3249-82) incubating overnight at 4°C, or 1:500 rabbit anti-α-catenin (Thermo, 71-1200), 1:1000 rabbit anti-caveolin 1 (Sigma, C4490), 1:1,000,000 rabbit anti-GroEL (Sigma, G6532), 1:1000 mouse anti-GAPDH (Developmental Studies Hybridoma Bank, DSHB-hGAPDH-2G7), 1:40,000 rabbit anti-keratin 8 (Abcam, Ab53280), 1:10,000 rabbit anti-keratin 18 (Abcam, ab668) 1:5000 goat anti-rabbit HRP (JacksonImmuno, 111-035-144), 1:5000 goat anti-rat HRP (JacksonImmuno, 112-035-003), and 1:5000 goat anti-mouse HRP (JacksonImmuno, 115035003) for 2 hours at room temperature. Western blots were developed, and signal was acquired by exposure to film. To determine band density, film was digitized using an Epson Perfection 4990 photo scanner and band intensity was measured using ImageJ.

Non-detergent sodium carbonate solution was used to extract lipid microdomains (Zhang et al., 2005 (link)). HK-2 cells were grown to near confluence in 100-mm² dishes. After two washes with ice-cold phosphate-buffered saline, two confluent dishes were scraped into 1 ml of 500 mM sodium carbonate, pH 11.0, in the presence of 1 mM PMSF and protease inhibitors cocktail. Homogenization was carried out with 10 strokes using a tight-fitting Dounce homogenizer, followed by three 20-sec bursts at 30% of maximal power to disrupt cellular membranes. The homogenates were adjusted to 45% sucrose by addition of 1 ml of 90% sucrose prepared in MBS [2-(N-morpholino) ethanesulfonic acid-buffered saline, 25 mM 2-(N-morpholino) ethanesulfonic acid, pH 6.5, and 0.15 M NaCl] and placed at the bottom of an ultracentrifuge tube. A discontinuous sucrose gradient (6 ml of 35% sucrose and 4 ml of 5% sucrose, both prepared in MBS) was formed above and centrifuged at 39,000 rpm for 20 h in an SW40 TI rotor (Beckman Instruments). A light-scattering band was observed at the 3–35% sucrose interface. Twelve 1-ml fractions were collected from the top of the tubes, and equal portion of each fraction was separated by SDS-PAGE for Western blot analysis as described above. The primary antibodies were goat anti-hKIM-1, rabbit anti-clathrin, rabbit anti-caveolin-1, and mouse anti-β-actin IgG (Sigma–Aldrich).