Fig. 7
β-alanine binding to GPER in dorsal root ganglion (DRG) neurons and microglia cells in vitro. A Representative immunoblotting analysis of the protein lysates of DRG neurons isolated from blank control, Vehicle, and β-alanine rats DRG tissues and heated at a series of temperatures from 42 to 72 °C for 3 min. Statistical regression (4 parameter logistic regressions, p < 0.05) of separate immunoblots generating the cellular thermal shift assay curves for the enhanced thermal stability of GPER by β-alanine. (B and C) Representative images (B) and quantifications (C, n = 6 sections from three rat DRGs) showed the GPER protein coexpressed in three different types of cell neuron, microglia and astrocytes. The corner image in the white square is the zoomed-in image of the area in the smaller white square. D The protocol of neuron, and microglial cell transfection with GPER-siRNA and β-alanine incubation. E Western blot images for GPER, iNOS, IL-1β, IL-6, and GABAα2 in the neurons and microglia. F Western blotting analysis diminished the protein expression of GPER, iNOS, IL-1β, IL-6, and GABAα2 (n = 3). Two-tailed unpaired Student’s t-test. ***p < 0.001 versus control group; ##p < 0.01 ###p < 0.001 versus NC-siRNA + β-alanine group; $$$p < 0.001 versus β-alanine