FIGURE 2:

Compartmentalization of ectopically expressed myc₇-Glut4 in undifferentiated preadipocytes. (A) Undifferentiated and differentiated cells expressing low levels of myc₇-Glut4 (LG), high levels of myc₇-Glut4 (HG), or both myc₇-Glut4 and sortilin-myc/His (GS) were homogenized, and total cell lysates were analyzed by Western blotting. Cellugyrin and glyceraldehyde 3-phosphate dehydrogenase were used as a loading control. (B) Quantification of myc₇-Glut4 signal shown in A, based on three independent experiments. *p < 0.05, **p < 0.01. (C) Undifferentiated LG, HG, and GS preadipocytes were homogenized and centrifuged at 27,000 × g for 35 min. Supernatants were pelleted by centrifugation at 200,000 × g for 90 min and analyzed by Western blotting along with aliquots of 27,000 × g pellets. Representative result of three independent experiments. (D) Undifferentiated HG and GS preadipocytes were homogenized and centrifuged at 27,000 × g for 35 min. Supernatants were analyzed by continuous sucrose gradient centrifugation for 55 min in a SW55 rotor at 48,000 rpm. The arrow indicates direction of sedimentation. Fractions with myc₇-Glut4 from HG and GS preadipocytes were transferred to the same membrane and processed simultaneously and therefore are directly comparable to each other. Representative result of at least four independent experiments.