FIG 3.

Cellular uptake of CLC, CLF-1, and CLC:CLF-1. (A) Untransfected and sorLA transfected HEK293 cells were incubated (37°C, 25 min) at 10 nM concentrations of the indicated ligands and then washed, fixed, and permeabilized. The cells were finally stained using mouse anti-His or rabbit anti-CLC as primary antibodies and Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit antibodies as secondary antibodies. (B) Colocalization of internalized CLC:CLF-1 and sorLA. sorLA transfected HEK293 cells were incubated with CLC:CLF-1 as described for panel A and subsequently stained with rabbit anti-CLC, mouse anti-sorLA, and matching secondary antibodies. (C) Colocalization of CLC:CLF-1 and endogenous CNTFRα (10 nM, 25 min). Following fixation the cells were stained with mouse anti-His, goat anti-CNTFRα, and appropriate secondary antibodies (Alexa Fluor 488-conjugated anti-mouse Ig and Alexa Fluor 568-conjugated anti-goat Ig).