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. Author manuscript; available in PMC: 2017 Aug 9.
Published in final edited form as: Cell Signal. 2015 Apr 6;27(7):1449–1456. doi: 10.1016/j.cellsig.2015.03.026

Figure 2. OX1R-mediated attenuation of KOR Gαi signaling is lost in the presence of the JNK inhibitor SP-600125.

Figure 2

(A): CHO-OX1R cells were serum starved in optimem for 4 h in the presence or absence of Gαq inhibitor UBO-QIC prior to treatment with the indicated concentrations of orexin. Cell lysates were collected and equal amounts of protein were run for western blotting. Phospho-JNK (pJNK) levels were analyzed using a rabbit anti-pJNK antibody (Cell Signaling) and loading was controlled using a mouse anti-actin antibody (Santa Cruz). (B): CHO-KOR cells were co-transfected with ‘GloSensor-22F’ cAMP sensor and HA-OX1R. After 48 h, cells were incubated with or without 20 μM SP-600125, PKI or H89 for 2 h. Cells were then pretreated or not with 500 nM orexin for 10 min prior to treatment with 2 μM forskolin (fsk) or 2 μM forskolin + 1 μM dynorphin (dyn) for 15 min, as indicated. Data is normalized to the forskolin signal in the absence of orexin pretreatment for each condition and error bars represent standard deviation from the mean of 3 separate experiments (* indicates p < 0.01).