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. 2016 May 31;6:27002. doi: 10.1038/srep27002

Figure 2. Analysis of the effect of GPR21 on the MAPKs, insulin signalling and glucose uptake.

Figure 2

(a) HEK293T cells transiently transfected with pCMV6-Entry or GPR21 were incubated with 50 nM GF109203X or an equal volume of the vehicle control, DMSO, for 16 h in serum free medium. (b) HEK293T cells transiently transfected with pCMV6-Entry or GPR21 were incubated with 10 μM U73122, 10 μM SP600125 or an equal volume of DMSO in serum free medium for 24 h. (c) HEK293T cells transiently transfected with pCMV6-Entry or GPR21 and (d) pCMV6-Entry or GPR21 coupled with Gα15/16, were incubated for 24 h in the presence or absence of 10% (v/v) FBS. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with antibodies against phospho-PKCδ Thr505, phospho-Erk Thr202/Tyr204, Erk, phospho-p38 Thr180/Tyr182, p38, phospho-JNK Thr183/Tyr185, JNK, myc and β-actin. Western blots are representative of two separate experiments. (e) HEK293T cells transiently transfected with pCMV6-Entry or GPR21 and (f) pCMV6-Entry or GPR21 coupled with Gα15/16, were incubated for 24 h in the presence or absence of 10% (v/v) FBS, then stimulated with 100 nM insulin for 1 h. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with antibodies against phospho-Insulin Receptor β Tyr1150/1151, Insulin Receptor β, phospho-IRS1 Tyr612, IRS1, phospho-Akt Ser473, Akt, phospho-AS160 Thr642, phospho-AS160 Ser588, AS160, myc and β-actin. Western blots are representative of three separate experiments. Alternatively, the consequential effect on glucose uptake was established by measuring cellular levels of [3H]-2-deoxyglucose in cells transiently transfected with (g) pCMV6-Entry or GPR21 and (h) pCMV6-Entry or GPR21 coupled with Gα15/16. Data presented as mean ± SEM are representative of two independent experiments performed in triplicate. One-way ANOVA with a post-hoc Tukey test conveys a significant increase in glucose uptake at p < 0.05* and p < 0.01**. A significant decrease in glucose uptake is denoted at p < 0.05#, p < 0.01## and p < 0.001###.