Figure 2. Depletion of both flotillin-1 and flotillin-2 does not inhibit PMA-dependent endocytosis of DAT in HEK293 cells.

(A) Summary of quantification of several experiments performed as in Fig. 1. HEK/CFP-HA-DAT cells were transfected with non-targeting siRNA (NT), SmartPool^™ (SP) to flotillin-2, siRNA to flotillin-1 (D6 or SP) or together flotillin-1 (D6) and flotillin-2 (SP). Cells were treated with 1 μM PMA or DMSO (veh) for 30 min. HA11 internalization assay was performed as described in “Methods” with Cy5 and Cy3 conjugated secondary antibodies detecting, respectively, surface and internalized HA11: CFP-HA-DAT complexes. Bars represent the mean values (+/−S.E.) of internalized/surface ratio (%) of HA11-DAT fluorescence normalized to this ratio in control (NT) cells treated with PMA.

(B) Representative example of western blot detection of flotilin-1 and flotillin-2 in HEK/CFP-HA-DAT cells treated as in A. Total cell lysates were equally divided and resolved on two parallel gels, then separately blotted with antibodies to flotillin-1, flotillin-2 and α-actinin (loading control). Flotillins were depleted on average by 73.4% in experiments presented in (A).

(C) Representative example of western blot detection of flotilin-1 and flotillin-2 in HEK/DAT cells expressing untagged DAT (HEK/DAT) cells treated as in A. Total cell lysates were equally divided and resolved on two parallel gels, then separately blotted with antibodies to flotillin-1, flotillin-2 and α-actinin (loading control). Cells from the same transfection were used in parallel experiment shown in Figure 3 with average depletion of flotillins by 76.5%.