Fig. 3
Association of folate transporters (PCFT, FBP, and RFC) proteins with lipid rafts in liver BLM. a Association of PCFT (54 kDa), FBP (27 kDa), and RFC (58 kDa) with DI fractions of liver BLM. Liver BLMVs were centrifuged for 30 min at 100,000g at 4 °C and suspended in MES buffer containing 50 mM MES (pH 6.5), 60 mM NaCl, 3 mM EGTA, 5 mM MgCl2, 1 % Triton X-100, and 1× complete protease inhibitor cocktail. Membrane vesicles were then incubated with MES buffer on a rotary shaker for 30 min at 4 °C. At the end of the incubation, BLMVs were centrifuged at 100,000g at 4 °C for 30 min, and supernatant was designated as DS fraction. The pellet was resuspended in buffer containing 15 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM EDTA, 1 mM DTT, 1 % Triton X-100, 0.1 % SDS, and 1× complete protease inhibitor cocktail and was designated as DI fraction. Proteins (50 μg) from DI and DS fractions were separated by electrophoresis on 10 % polyacrylamide gel and then analyzed by Western blotting for PCFT, FBP, and RFC expression. b, d The liver BLM was subjected to floatation on Optiprep density gradients, and fractions were collected from top of the gradients (fractions 1–4 represent detergent-resistant membrane). Fractions were separated by electrophoresis and analyzed by Western blotting using a anti-PCFT (54 kDa) and b RFC (58 kDa) antibodies. c, e Blots were scanned, and the intensity of bands was determined by densitometric analysis. Data are mean ± SD of 4 separate experiments. The represented blots shown for PCFT and RFC expression as, lane 1–5: Control; lane 1′–5′: ethanol fed. *p < 0.05; **p < 0.01; ***p < 0.001 versus control