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. Author manuscript; available in PMC: 2006 Nov 1.

Published in final edited form as: Mol Endocrinol. 2006 Jun 27;20(11):2931–2945. doi: 10.1210/me.2006-0138

Confocal imaging of the myc-hLHR-wt and mutants thereof 293T cells were transiently transfected with the indicated myc-hLHR constructs without (left panels) or with cathepsin D-GFP (right panels). The transfected cells were washed and incubated without (-hCG) or hCG (2.5 nM, Kd = 1-3 nM) for 20 min at 37°C (+ hCG, t = 0) and further processed as described in Materials and Methods. Another group of cells was incubated with hCG (52 nM) at 37°C for 20 min and then, after removal of the receptor-bound hormone they were further incubated without hCG for another 2 h at 37°C (+hCG, t = 2h) to allow for processing of the internalized hormone. The cells transfected with the receptor only (left panels) were also incubated for 2.5 min with Alexa488 conjugated-transferrin and then treated with an isotonic acid buffer to release the surface-bound transferrin while leaving the internalized transferrin in the early endosomes. The receptors (in red) were visualized using an anti-myc monoclonal antibody (9E10) and a CY5™-conjugated anti-mouse antibody. Alexa488 conjugated transferrin and cathepsin D-GFP are shown in green and colocalized compartment are shown in yellow. The cells were observed and analyzed using a Zeiss confocal microscope.

Confocal imaging of the myc-hLHR-wt and mutants thereof 293T cells were transiently transfected with the indicated myc-hLHR constructs without (left panels) or with cathepsin D-GFP (right panels). The transfected cells were washed and incubated without (-hCG) or hCG (2.5 nM, Kd = 1-3 nM) for 20 min at 37°C (+ hCG, t = 0) and further processed as described in Materials and Methods. Another group of cells was incubated with hCG (52 nM) at 37°C for 20 min and then, after removal of the receptor-bound hormone they were further incubated without hCG for another 2 h at 37°C (+hCG, t = 2h) to allow for processing of the internalized hormone. The cells transfected with the receptor only (left panels) were also incubated for 2.5 min with Alexa488 conjugated-transferrin and then treated with an isotonic acid buffer to release the surface-bound transferrin while leaving the internalized transferrin in the early endosomes. The receptors (in red) were visualized using an anti-myc monoclonal antibody (9E10) and a CY5™-conjugated anti-mouse antibody. Alexa488 conjugated transferrin and cathepsin D-GFP are shown in green and colocalized compartment are shown in yellow. The cells were observed and analyzed using a Zeiss confocal microscope.