Effects of pathway inhibitors on signalling responses to mINSL5 in CHO‐RXFP4 cells. CHO‐RXFP4 cells were pretreated with the Gαi/o inhibitor (PTX, 100 ng•mL−1), MEK inhibitor (U0126, 10 μM), Src family tyrosine kinase inhibitor (PP2, 10 μM), PI3K inhibitor (LY294002, 10 μM) or the mTOR complex inhibitors (KU0063794, 1 μM; rapamycin, 100 nM) followed by INSL5 (100 nM). Pathways examined were (A) p‐ERK1/2, (B) Akt p‐Ser473 , (C) Akt p‐Thr308 and (D) p‐S6RP. Cells were treated with inhibitors for 30 min before and then during mINSL5 stimulation, except for the PTX treatment where cells were exposed for 18 h. Results are quantified as fold change in fluorescence over that of the vehicle treatment in the control group. Bars represent mean ± SEM of experiments [n = 5 (PTX, LY294002, KU0063794, rapamycin), n = 6 (PP2, U0126), n = 10 (Control)]. *P < 0.05; significantly different from the mINSL5 response in the control group; repeated‐measures two‐way ANOVA followed by Dunnett's multiple comparisons test. ns, non‐significant.