Fig 1: FRET intensity in T-REx 293 overexpressing system between the receptors labeled with Snap-Tb (donor) and Snap-Green (acceptor) presented on the surface of the cells (a, b 5-HT1A; receptor cells, c, d mGlu4 receptor cells). The signal was measured at the specific emission of 520 nm for the acceptor and at 490 nm for the donor after excitation at 340 nm. a and c FRET intensity in single- and b and d in double-labeled cells. The ratio of FRET and Snap-Tb × 104 in double-labeled cells treated with agonist (R)-(+)-8-OH DPAT (b) or l-glutamate (d). e and f Demonstrate the interaction between mGlu4 and 5-HT1A receptors tagged with Snap-Tb and Halo-Alexa 488, respectively, presented on the surface of the T-REx 293 cell line. e A significant (p < 0.001) increase in FRET fluorescence was observed in the double-labeled cells. f The effect of glutamate and/or (R)-(+)-8-OH DPAT on the ratio of FRET and Snap-Tb/Halo-Alexa 488 × 104. ONE-way ANOVA **p < 0.01, ***p < 0.001
Fig 2: Effect of sulfasalazine (SSZ) at a concentration of 100 µM on receptors activity in cAMP accumulation assay. a Sulfasalazine treatment significantly enhanced 5-HT1A activity in presence of mGlu4 receptors (filled triangle) in comparison to cells without sulfasalazine administration (filed square) where expression of mGluR4 induced by tetracycline inhibited the serotonin receptor function. b Sulfasalazine treatment shifted the dose–response curve of glutamate to the left
Fig 3: Effect of the coexpression of 5-HT1A and mGlu4 in the T-REx 293 cell line. The expression of mGlu4R was induced by tetracycline administration (+ Tet). a Effect of L-glutamate on forskolin-stimulated cAMP accumulation in cells with or without tetracycline induction. b Effect of (R)-(+)-8-OH DAPT on forskolin-stimulated cAMP accumulation in cells with or without tetracycline induction
Fig 4: a The curve of representative effects of increased concentrations of L-Glu on cAMP accumulation with or without the presence of VU0155041, an mGlu4 receptor PAM in cells overexpressing human GRM4 fused with SNAP. The results show mean ± SD from three replicates. b The 5-HT1A agonist (R)-(+)-8-OH DPAT induced inhibition of 5 µM forskolin-stimulated cAMP production in T-REx 293 cells overexpressing human 5HT1A receptor tagged with Snap protein. The effect of the agonist was abolished by the addition of an 5-HT1A antagonist WAY100135
Fig 5: mGlu4 and 5-HT1A receptors co-localize in different regions of the mouse brain. The sections of the brain labeled with anti-mGlu4 receptor primary antibody and detected with Alexa488 donkey anti-rabbit secondary antibody (green) are shown in the first column. The sections labeled with anti-5HT1A receptor (5-HT1AR) primary antibody and detected with Alexa555 donkey anti-goat secondary antibody (red) are shown in the second column. The nuclei labeled with DAPI (blue) are shown in the third column. Figure 6 also demonstrated the specificity of antibody anti 5-HT1a and anti-mGluR4 used for colocalization staining. Both demonstrated localization in well known and described region of expression like: CA1 piramidal cells layer. In merge arrows indicate some of cells and regions stained with DAPI only where antibodies did not bind to antigens. Scale bars = 20 µm
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