Figure 2

WIN55,212-2 promotes internalization of CB1Rs in cultured astrocytes. Labeling of surface CB1Rs in living astrocytes: astrocytes were incubated with WIN55,212-2 for 1 h, then CB1R antibodies (1:500) labeled with Alexa-488 were added to the cultures for 30 min. The antibodies were washed out and the tissue was fixed. The CB1R was labeled by antibodies targeting the first external loop (1L) or the N-terminus (Nt). Anti-CB1R Nt was used in A1, B1, A3, and B3; anti-CB1R 1EL was used in A2 and B2. Green = CB1R, blue = DAPI; magnification = 60×. Labeling of internalized CB1Rs in fixed astrocytes: astrocytes were first incubated with WIN55,212-2 for 1 h. Afterwards they were fixed and incubated with a solution containing 0.1% Triton X100. The CB1R antisera were added in this solution and incubated for 2 h. Anti-CB1R Nt was used in C1, D1, and E1; anti-CB1R 1EL was used in C2, D2, and E2. Details in Methods and Results.