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Table 1 Effect of cholesterol depletion on distribution of marker proteins during sucrose gradient centrifugation

From: Cholesterol Depletion Blocks Redistribution of Lipid Raft Components and Insulin-Mimetic Signaling by Glimepiride and Phosphoinositolglycans in Rat Adipocytes

 

Control

2 mM mβCD

10 mM mβCD

fr. no.

4–5

8–9

12–15

4–5

8–9

12–15

4–5

8–9

12–15

cav-1

1.00 ± 0.03

0.08 ± 0.02

0.03

1.13 ± 0.27

0.16 ± 0.04

0.05 ± 0.02

0.91 ± 0.10

0.19 ± 0.07

0.13 ± 0.03

cav-2

1.00 ± 0.09

0.11 ± 0.04

0.05 ± 0.01

1.21 ± 0.07

0.09 ± 0.07

0.07 ± 0.02

0.85 ± 0.09

0.24 ± 0.10

0.17 ± 0.04

pp59Lyn

1.00 ± 0.12

0.76 ± 0.11

0.01

0.89 ± 0.06

0.92 ± 0.14

0.04

0.20 ± 0.09

1.72 ± 0.19

0.08 ± 0.02

5′-Nuc

1.00 ± 0.15

0.13 ± 0.04

0.07 ± 0.02

0.84 ± 0.11

0.23 ± 0.03

0.03 ± 0.02

0.40 ± 0.08

0.69 ± 0.14

0.12 ± 0.03

Gce1

1.00 ± 0.19

0.11 ± 0.02

0.04 ± 0.01

0.79 ± 0.12

0.21 ± 0.06

0.06

0.49 ± 0.07

0.55 ± 0.10

0.09 ± 0.02

IRβ

1.00 ± 0.11

0.89 ± 0.10

7.89 ± 0.78

0.70 ± 0.13

1.43 ± 0.25

8.33 ± 0.92

0.45 ± 0.09

1.72 ± 0.29

9.03 ± 0.85

GLUT4

1.00 ± 0.19

0.54 ± 0.08

8.72 ± 1.11

0.92 ± 0.15

1.05 ± 0.18

9.44 ± 1.06

0.66 ± 0.08

2.03 ± 0.39

10.27 ± 1.54

CIR

1.00 ± 0.14

0.24 ± 0.09

0.02

0.54 ± 0.06

0.32 ± 0.11

0.01

0.22 ± 0.11

0.81 ± 0.13

0.06 ± 0.01

  1. Isolated rat adipocytes were incubated (50 min, 30°C) in the absence or presence of mβCD. DIGs were prepared and subjected to sucrose gradient centrifugation. Fractions 4–5, 8–9, and 12–15 were immunoblotted for caveolin-1/2 (cav-1/2), pp59Lyn, IRβ and GLUT4, assayed for 5′-nucleotidase (5′-Nuc) enzymic activity, photoaffinity labeled for Gce1, and labeled with N-ethyl[2,3-14C] maleimide for CIR. Quantitative evaluations (chemi- and phosphorimages, enzymatic assay) represent the mean ± SD of three to four independent adipocyte preparations with incubations in triplicate each (fractions 4–5 in the absence of mβCD set at 1). The total amount of each protein recovered in the three fraction pools of mβCD-treated adipocytes was 88–113% (2mM) and 81–93% (10 mM) of that of untreated cells.
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Molecular Medicine

ISSN: 1528-3658