Supplementary Figure 2: DA biosynthesis and degradation in the NAc of Vav2 mutant mice are not significantly altered.

a, Western analysis of VTA tissue lysates from adult Vav2-null mouse brain, in which rabbit monoclonal anti-Vav2 antibody was used, shows the absence of Vav2 protein. The experiments were repeated at least twice. b, Quantitative data of a. There is no significant difference in the levels of TH and VMAT2 protein expression between genotypes. unpaired two-tailed t-test. (n = 3 per group); t₍₄₎ = 1.460, P = 0.2182. *P = 0.2182. c-e, Western analysis of phosphor-TH (ser 40) expression in the NAc (c, d) or VTA (c, e) of Vav2 mutant and WT mice. d, e, Quantitative data of (c). unpaired two-tailed t-test. (d, n = 6 mice per group, t₍₁₀₎ = 0.8557, P = 0.4122, e, n = 5 mice per group, t₍₈₎ = 0.3065, P = 0.7670). f, The accumulation of l-dopa in the NAc was measured in the animals exposed to NSD-1015, an aromatic acid decarboxylase (AADC) inhibitor, using HPLC. Lower levels of L-dopa were seen in the NAc of Vav2 mutant mice, suggesting that TH enzymatic activity was markedly inhibited. unpaired two-tailed t-test. (n = 5 mice per group); t₍₈₎ = 2.400, *P = 0.0432. g, h, qPCR analysis of mRNA expression of MAO-A (g) and MAO-B (h), which are responsible for enzymatic degradation of 5-HT, NE and DA in the VTA and NAc of Vav2 mutant and WT mice. unpaired two-tailed t-test. (n = 3 mice). (g, t₍₄₎ = 0.3998, P = 0.7097 for VTA, t₍₄₎ = 0.06329, P = 0.9526 for NAc; h, t₍₄₎ = 2.946, P = 0.0421 for VTA, t₍₄₎ = 2.382, P = 0.0758 for NAc). i, qPCR analysis of MAO-A and Vav2 mRNA expression in MN9D dopaminergic cell line transfected with shVav2 #2. unpaired two-tailed t-test. (n = 3 independent experiments). t₍₄₎ = 35.55, *P < 0.0001 for Vav2, t₍₄₎ = 0.0, *P > 0.9999 for MAO-A. j, Measurement of enzymatic activities of MAO-A were performed in the VTA and NAc of Vav2 mutant and WT mice. unpaired two-tailed t-test. (n = 3 mice per group). t₍₄₎ = 0.1290, P = 0.9036 for VTA, t₍₄₎ = 1.202, P =0.2958 for NAc. k, Immunoblotting reveals that the levels of VMAT2 expression were significantly altered in the striatum of Vav2 mutant mice compared with wild-type counterparts. l, Quantitative data shown in k. unpaired two-tailed t-test. (n = 6 mice per group). t₍₁₀₎ = 0.02854, P = 0.9778. m, Full-length gel showing immunoblotting analysis of phosphor-Thr53-DAT expression. Their expression levels were not significantly changed between genotypes in various brain regions. n, Quantitative data shown in l. unpaired two-tailed t-test. (n = 5 mice). t₍₈₎ = 1.290, P = 0.2331 for NAc, t₍₈₎ = 1.849, P = 0.1016 for dSTR, t₍₈₎ = 1.146, P = 0.2847 for VTA. Error bars represent s.e.m. n.s., not significant. k shows cropped blot images from their corresponding full-length blot presented in Supplementary Figure 12.