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. 2019 Mar 5;8:e40234. doi: 10.7554/eLife.40234

Figure 2. Ceramide stimulates RAT of CCR5.

(A) Schematic illustration of the effect of RAT on O-linked glycosylation of CCR5. (B) SV589 cells transfected with pCCR5-myc were treated with 8 μM C6-ceramide for the indicated time followed by immunoblot analysis with anti-Myc. (C) SV589 cells transfected with the indicated plasmid were treated with or without 8 μM C6-ceramide for 16 hr followed by immunoblot analysis with anti-myc. (D) HEK293 cells transfected with pSNAP-CCR5 were treated with 8 μM C6-ceramide for 16 hr, labeled with a cell-impermeable fluorescent substrate for SNAP-tag, and quantified the labeling reaction through a fluorimeter. Results are reported as mean ±S.E. from triplicate incubations of a typical experiment. (E) Schematic illustration of the effect of RAT on cell surface labeling of extracellular cysteine residues in wildtype and the topology reporter CCR5. (F and G) SV589 cells transfected with Myc-tagged wildtype or topology reporter CCR5 were treated with 8 μM C6-ceramide for 16 hr. After cell surface labeling of extracellular cysteine residues with biotin, cell lysates were precipitated with streptavidin beads. Equal fractions of whole cell lysate (W), pellet (P) and supernatant (S) were subjected to immunoblot analysis with anti-Myc.

Figure 2.

Figure 2—figure supplement 1. Mutations bocking glycosylation do not affect RAT of CCR5.

Figure 2—figure supplement 1.

Localization of the Myc-tag fused at the C-terminus of CCR5(S6A, S7A, T16A, S17A) was analyzed as described in Figure 1. Scale bar = 50 μm.

Figure 2—figure supplement 2. Ceramide treatment does not affect expression of SNAP-CCR5.

Figure 2—figure supplement 2.

HEK293 cells were setup, transfected, and treated as described in Figure 2D. The amount of transfected SNAP-tagged CCR5 was analyzed by immunoblot analysis with anti-SNAP.