Fig. 1

a Primary brainstem and spinal cord neurons were treated with 10 µM purmorphamine at the indicated times and glycine transport rates were measured using [³H]-glycine transport assays. Glycine transport shown is normalized against control conditions. ****p (PMM 16 h) < 0.0001, ****p (PMM 24 h) < 0.0001, using Dunn’s multiple comparisons test, Number of experiments n (Veh & PMM 2 h) = 5, n = (Veh & PMM 8 h) = 5, n (Veh & PMM 16 h) = 11, n (Veh & PMM 24 h) = 6. b Representative immunoblot of primary brainstem and spinal cord neuronal cultures. Cells were treated with 10 µM purmorphamine for the times indicated. Tubulin is used as protein loading control. Veh: DMSO. c Quantification is shown normalized to the corrected signal in the control in each case (Veh). ****p (PMM 16 h) < 0.0001, ***p (PMM 24 h) = 0.0002, using Dunn’s multiple comparisons test, number of experiments n (Veh & PMM 2 h) = 4, n = (Veh & PMM 8 h) = 8, n (Veh & PMM 16 h) = 10, n (Veh & PMM 24 h) = 5. d Neurons were treated with the vehicle alone or with 10 µM purmorphamine and the expression of the following GlyT2-associated/related proteins was assessed: α3NKA, CRMP5, and syntaxin1A (STX1A). Tubulin is used as protein loading control. e Box plots show quantification of protein expression changes during 8 and 16 h PMM treatment measured by western blots performed as in Fig. 1d. Data are normalized to the corrected signal in the control in each case (Veh). ^nsp (αNKA: PMM 8 h) = 0.7260, ^nsp (αNKA: PMM 24 h) > 0.9999, ^nsp (CRMP5: PMM 8 h) > 0.9999, ^nsp (CRMP5: PMM 24 h) > 0.9999, ^nsp (STX1A: PMM 8 h) = 0.7260, ^nsp (STX1A: PMM 24 h) > 0.9999, using Dunn’s multiple comparisons test, number of experiments n = 3. PMM purmorphamine