Xu Y , Li L , Zheng J , Wang M , Jiang B , Zhai Y , Lu L , Zhang C , Kuang Z , Yang X , Jin LN , Lin G , Zhang C .

	Figure 1. Protein alignment and phylogenetic analysis of Xenopus laevis MC3Rs. (A) Sequence alignments of xlMC3Rs (Xenopus laevis MC3R.L, XP_018090440.1; Xenopus laevis MC3R.S, XP_018093682.1) and other MC3Rs from human (NP_063941.3), mouse (NP_032587.1), Norway rat (NP_001020441.3), pig (NP_001116609.1), cattle (XP_010809919.1), chicken (XP_004947293.1), common wall lizard (XP_028591076.1), green sea turtle (XP_007059824.1), Xenopus tropicalis (XP_002935436.1), Microcaecilia unicolor (XP_030069319.1), two-lined caecilian (XP_029467773.1), zebrafish (NP_851303.2), elephant shark (XP_007883784.1), and sea lamprey (ABB36647.1). The blue, red, and yellow represent a homology over 50%, 75%, and 100%, respectively. (B) Dendrogram of MC3Rs was generated by the NJ analysis with Molecular Evolutionary Genetics Analysis (MEGA) software.
	Figure 2. Synteny analysis of Xenopus laevis MC3Rs. Synteny mapping of MC3Rs with Callorhinchus milii (elephant shark), Danio rerio (zebrafish), Rhinatrema bivittatum (two-lined caecilian), Microcaecilia unicolor, Xenopus tropicalis, Xenopus laevis, Podarcis muralis (common wall lizard), Gallus gallus (chicken), Mus musculus (house mouse), and Homo sapiens (human). Genes in blue represent positional genomic conservatism during evolution among multiple species.
	Figure 3. mRNA expression of xlmc3r.L/S. Expression profiles of xlmc3r.L (A) and xlmc3r.S (B) in multiple tissues from an adult male Xenopus laevis. The relative expression was normalized to the housekeeping gene xlactb.L. Data were plotted as mean ± s.e.m. of three independent experiments. Br, brain; He, heart; St, stomach; Pan, pancreas; Sp, spleen; Lv, liver; Tes, testis; fat; Kd, kidney.
	Figure 4. Protein interaction of xlMRAPs and xlMC3Rs in vitro. (A) Co-immunoprecipitation of the 3×HA-xlMC3R.L and 2×Flag-xlMRAPs. (B) Co-immunoprecipitation of 3×HA-xlMC3R.S and 2×Flag-xlMRAPs. The bimolecular fluorescence complementation assay showed the co-localizations of (C) xlMC3R.L or (D) xlMC3R.S and xlMRAPs. Venus fluorescence imaging (green) and 2-Flag (red) exhibited protein complex of xlMRAP1.L (or xlMRAP2.L/S) and xlMC3Rs. Nuclei were in blue (DAPI). Scale bar = 10 μm.
	Figure 5. Modulation of xlMC3R.L/S signaling by xlMRAPs. (A, B, C, D, E, and F) Dose–response curves of agonist (α-MSH) (0 M, 10−11 to 10−7 M) stimulated cAMP production of xlMC3R.L with different ratio of (A) xlMRAP2.L, (B) xlMRAP2.S, and (C) xlMRAP1.L. Ligand stimulation of xlMC3R.S was also modulated by (D) xlMRAP2.L, (E) xlMRAP2.S, and (F) xlMRAP1.L. All data of ligand stimulation were normalized to the maxima of 1:0, 1:1, 1:3, and 1:6 curves in the ligand stimulation assay. Data were plotted as the mean ± s.e.m. of three independent experiments performed in triplicate. (G, H, I, J, K, and L) The antagonistic ability of SHU9119 (10−11 to 10−6 M) in the presence of α-MSH (EC80) induced the alteration of xlMC3R.L signaling with different amounts of (G) xlMRAP2.L, (H) xlMRAP2.S, and (I) xlMRAP1.L. Antagonistic ability of SHU9119 (10−11 to 10−6 M) in the presence of α-MSH (EC80) induced the alteration of xlMC3R.S signaling was also regulated by (J) xlMRAP2.L, (K) xlMRAP2.S, and (L) xlMRAP1.L. All data of antagonistic ability were normalized to the maxima of 1:0, 1:1, 1:3, and 1:6 curves. Data were plotted as the mean ± s.e.m. of three independent experiments performed in triplicate.
	Figure 6. Alteration of the surface expression of xlMC3R.L and xlMC3R.S by xlMRAPs at ratio of 1:0, 1:1, 1:3, and 1:6. Surface expression of the N-terminally 3×HA tagged xlMC3R.L in the presence of (A) the N-terminally 2×Flag tagged xlMRAP2.L, (B) the N-terminally 2×Flag tagged xlMRAP2.S, or (C) the N-terminally 2×Flag tagged xlMRAP1.L. Whole expression of the N-terminally 2×Flag tagged xlMRAP2.L (D and J), the N-terminally 2×Flag tagged xlMRAP2.S (E and K), and the N-terminally 2×Flag tagged xlMRAP1.L (F and L). Surface expression of the N-terminally 3×HA tagged xlMC3R.S in the presence of (G) the N-terminally 2×Flag tagged xlMRAP2.L, (H) the N-terminally 2×Flag tagged xlMRAP2.S, or (I) the N-terminally 2×Flag tagged xlMRAP1.L. Each receptor/accessory protein expression level was shown as fold difference compared to HEK293T cells expressing 3×HA-xlMC3R alone. Data were plotted as the mean ± s.e.m. of three independent experiments performed in triplicate, not significant [ns], ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 one-way ANOVA.

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