Figure 4. Biochemical enrichment of... | Rockefeller University Press

Figure 4.

$Figure 4. Biochemical enrichment of CLICs. (A) 3T3-GPI cells were subjected to density fractionation as described in Materials and methods. Western blots of membrane markers are shown. Within the first gradient (left) anti-GFP–, Cav1-, and Grp78-positive membranes are present in Fraction 1.2, highlighted by outline. CHC-, GM130-, and TfR-positive membranes are below the detection limit within this fraction. Within the second gradient, anti-GFP–positive membranes concentrate within Fraction 2.8, highlighted by outline. This represnts a yield of 9.6 ± 0.2% of anti-GFP–positive membranes. Cav1 concentrates in Fraction 2.6 and Grp78 in Fraction 2.10. (B) Fraction 2.8 was fixed and visualized by EM. Structures within Fraction 2.8 share similar profiles to CLICs seen within intact cells. Bar, 200 nm. (C) High resolution electron micrographs of Fraction 2.8 structures, providing examples of vesiculation (arrows) and a spherical bulb connected to tubular extension (arrowheads). Bar, 100 nm. (D) NIH3T3 cells were incubated with CTxB-HRP before fractionation and the DAB reaction was performed on Fraction 2.8. Bar, 200 nm. (E) NIH3T3 cells transfected with Cdc42-WT or -DN were incubated with CTxB before fractionation. Western blots were probed with anti-CTxB. 3T3-GPI cells were treated or not with mβCD before incubation with anti-GFP antibodies. Western blots of fractions were probed with anti–Rb-HRP. (F) Western blots of fractions from cells transfected with Flot1-HA, GRAF1-myc, or left untransfected. Fractions were probed with anti-HA, -myc, -dynamin, -Flotillin1, or -GRAF1 antibodies as appropriate. (G) Fixed sampled from F of Fraction 2.8 were labeled for anti-HA, anti-myc, endogenous Cav1, or dynamin. Structures labeled with Flotillin-1-HA, GRAF1-myc, and dynamin (arrows) or Cav-1 (arrowheads) are shown. Bars, 200 nm.$

Biochemical enrichment of CLICs. (A) 3T3-GPI cells were subjected to density fractionation as described in Materials and methods. Western blots of membrane markers are shown. Within the first gradient (left) anti-GFP–, Cav1-, and Grp78-positive membranes are present in Fraction 1.2, highlighted by outline. CHC-, GM130-, and TfR-positive membranes are below the detection limit within this fraction. Within the second gradient, anti-GFP–positive membranes concentrate within Fraction 2.8, highlighted by outline. This represnts a yield of 9.6 ± 0.2% of anti-GFP–positive membranes. Cav1 concentrates in Fraction 2.6 and Grp78 in Fraction 2.10. (B) Fraction 2.8 was fixed and visualized by EM. Structures within Fraction 2.8 share similar profiles to CLICs seen within intact cells. Bar, 200 nm. (C) High resolution electron micrographs of Fraction 2.8 structures, providing examples of vesiculation (arrows) and a spherical bulb connected to tubular extension (arrowheads). Bar, 100 nm. (D) NIH3T3 cells were incubated with CTxB-HRP before fractionation and the DAB reaction was performed on Fraction 2.8. Bar, 200 nm. (E) NIH3T3 cells transfected with Cdc42-WT or -DN were incubated with CTxB before fractionation. Western blots were probed with anti-CTxB. 3T3-GPI cells were treated or not with mβCD before incubation with anti-GFP antibodies. Western blots of fractions were probed with anti–Rb-HRP. (F) Western blots of fractions from cells transfected with Flot1-HA, GRAF1-myc, or left untransfected. Fractions were probed with anti-HA, -myc, -dynamin, -Flotillin1, or -GRAF1 antibodies as appropriate. (G) Fixed sampled from F of Fraction 2.8 were labeled for anti-HA, anti-myc, endogenous Cav1, or dynamin. Structures labeled with Flotillin-1-HA, GRAF1-myc, and dynamin (arrows) or Cav-1 (arrowheads) are shown. Bars, 200 nm.