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. 2016 Jul 18;7:96. doi: 10.3389/fendo.2016.00096

Figure 7.

Figure 7

Stability of MRAP and MC2 receptor. (A) CHO cells stably expressing Flag-tagged MRAP were transfected with either GFP or HA-tagged MC2 receptor and then incubated for 4 h with no drug, 10 μg/ml cycloheximide (CHX), 50 μM MG132, or both. Cell lysates were run on SDS-PAGE and blotted for either (upper) MC2 receptor or (lower) MRAP. Brackets on the right show glycosylated MC2 receptor and core and non-glycosylated receptor. (B,D) CHO cells stably expressing Flag-tagged MRAP were incubated with 10 μg/ml cycloheximide (CHX), 50 μM MG132, or both for (B) times shown or (D) 1 h. In (B), cells were lysed and immunoprecipitation with anti-Flag antibody performed whereas in (D) cells were incubated for 15 min with1:1000 anti-Flag antibody and washed extensively before lysis and collection of Flag-labeled proteins on Protein A/G beads to isolate cell surface MRAP. (C) CHO and OS3 adrenal cells were transiently transfected with MRAP or MC2 receptor and MRAP, then incubated with 100 μg/ml cycloheximide for the times shown, lysed and blotted for MRAP.