Notes

1. L1 cells are a continuous substrain of 3T3 (Swiss albino) cells developed through clonal isolation. Cells undergo a preadipose-to-adipose-like conversion as they progress from a rapidly dividing to a confluent and contact inhibited state.

2. The purchased BCSS is collected from formula-fed calves, which produce exceptionally high levels of transferrin. It is supplemented with iron to load serum transferrin levels to physiological levels. BCSS is sterile filtered using three sequential 100-nm (0.1-^m) pore-size rated filters. This filtration system virtually eliminates contaminating mycoplasmas from the serum.

3. The purchased FBS is filtered through multiple 40-nm (0.04-mm) pore-size rated filters, which are the most retentive filters used in commercial FBS production. This highly retentive filtration method has been shown to significantly reduce the level of virus particles as small as 60 nm.

4. All media is prepared by filtration (0.2 im). As recommended by HyClone, we do not heat inactivate the serum products.

5. Cells should be split every 4 d. They can be passaged for approx eight times or until they start to grow at a much faster rate as evidenced by the cell counts. Plating rec-

Fig. 1. The subcellular fractionation technique. All steps are performed at 4°C. See text for abbreviations and details.

ommendations: T-150 flask, 100,000 cells; 150-mm dish, 400,000 cells; 100-mm dish, 200,000 cells; 60-mm dish, 100,000 cells; 35-mm fish, 50,000 cells.

6. Adipocytes at 10-12 d after withdrawal from differentiation media are routinely used, because at this time of adipocyte development, the mRNA and protein levels of GLUT-4 (14) together with the glucose-transport response to insulin (15) have reached a steady state.

7. Protease inhibitors should be added to ice-cold HES buffer just prior to use.

8. The pellet containing the HDM fraction is easily dislodged when removing the supernatant. Optimal recovery of the HDM pellet and LDM-containing supernatant can be achieved by carefully pipetting the supernatant out from the opposite side of the pellet while slightly tilting the tube with the pellet facing up. Because the protein yield of the LDM fraction is usually more than sufficient to perform electrophoresis and Western blotting analysis on, it is not necessary to obtain the entire supernatant, thus avoiding the contamination of the LDM fraction with the HDM fraction. The HDM pellet can be washed one or more times by resuspending in HES buffer and repeating the 41,000g/20-min centrifugation step.

9. The approximate total protein (ig) recovered in the HDM, LDM, CYT and PM fractions are 300, 1000, 5000, and 3000, respectively.

Fig. 2. Effect of insulin and osmotic shock on the subcellular distribution of GLUT-4. Differentiated 3T3-L1 adipocytes were stimulated without (C, lanes 1 and 4) or with 100 nM insulin (I, lanes 2 and 5), or with 600 mM sorbitol (S, lanes 3 and 6) for 30 min, and subcellular membrane fractions were obtained by differential centrifugation. To detect GLUT-4 protein, only 1 ^g of plasma membrane protein (PM, lanes 1-3) and low-density microsomal protein (LDM, lanes 4-6) were separated by SDS-PAGE and transferred to nitrocellulose membranes.

Fig. 2. Effect of insulin and osmotic shock on the subcellular distribution of GLUT-4. Differentiated 3T3-L1 adipocytes were stimulated without (C, lanes 1 and 4) or with 100 nM insulin (I, lanes 2 and 5), or with 600 mM sorbitol (S, lanes 3 and 6) for 30 min, and subcellular membrane fractions were obtained by differential centrifugation. To detect GLUT-4 protein, only 1 ^g of plasma membrane protein (PM, lanes 1-3) and low-density microsomal protein (LDM, lanes 4-6) were separated by SDS-PAGE and transferred to nitrocellulose membranes.

10. The purity of the fractions can be assessed with marker proteins [e.g., Ras (PM marker), GLUT-4 (LDM marker for basal preparations), cytochrome-c oxidase (for endoplasmic reticulum), and several others, as described previously (16).

11. Using the "sheet assay," we previously reported that, similar to insulin, osmotic shock of 3T3-L1 adipocytes stimulates an increase in the level of PM localized GLUT-4 (18). As shown in Fig. 2, stimulation of 3T3-L1 adipocytes with insulin-or sorbitol-induced osmotic shock results in a loss of GLUT-4 protein in the LDM fraction with an increase in the amount of GLUT-4 protein in the PM fraction. The decrease of the transporter in the LDM fraction concomitant with its recruitment to the PM fraction is characteristic of GLUT-4 translocation.

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  • can tubulin be used for checking purity of ldm and pm fractions?
    can tubulin be used for checking purity of ldm and pm fractions?
    7 years ago

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