Fig. 5. NGF expression in macrophages was reduced in Snx25^+/− mice.

a, Representative immunoblot showing NGF expression in the hind paw skin of WT and Snx25^+/− mice. The graph shows semi-quantitative analyses of the immunoblots (WT, n = 4; Snx25^+/−, n = 4). P = 0.002. b, Representative immunoblot showing NGF levels in the hind paw skin of WT and Snx25^+/− mice 30 min after formalin injection and semi-quantitative analyses of NGF levels in the ipsilateral (ipsi) hind paw skin of WT (n = 3) and Snx25^+/− mice (n = 3). P = 0.015. Cont, contralateral. c, Confocal microscopy of the hind paw skin immunolabeled for NGF and MHC-II, F4/80 and Iba1 (macrophage markers) and CD117 (mast cell marker) in WT mice. Arrowheads denoted double-labeled cells. Representative of three independent experiments. Scale bar, 50 μm. d, Immunoblot of NGF in BMDMs of WT and Snx25^+/− mice, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) content and analyzed semi-quantitatively (WT, n = 4; Snx25^+/−, n = 5, P = 0.015). e, Confocal microscopic images of sciatic nerve sections immunolabeled for TrkA at 8 h after nerve ligation (arrows indicate ligation site) in WT and Snx25^+/− mice (left), magnified views of the boxed areas in the corresponding left panels (middle) and semi-quantitative analysis of TrkA accumulation on the distal side of the nerve ligature (right) (n = 4 sciatic nerve sections from three different mice, P = 0.008). Representative of two independent experiments. Scale bar, 200 μm. f, Expression profiles of Snx25 and Ngf mRNA in BMDMs of WT and Snx25^+/− mice (WT, n = 6; Snx25^+/−, n = 6; Snx25, P = 0.049; Ngf, P = 0.085). g, Ngf mRNA quantified by RT–qPCR in BMDMs transfected with either Snx25 siRNA (siSnx25, n = 3) or scramble siRNA (siCtr, n = 3; P = 0.0085). h, Confocal microscopy of YFP-labeled BMDMs derived from Snx25^Cx3cr1-cKO;Ai32^Tg/+ mice without (top) or with (bottom, 1 μM, 7–8 d) 4-OHT treatment. Representative of three independent experiments. Scale bar, 100 μm. i, Flow cytometry of PI and YFP expression in BMDMs cultured from Snx25^Cx3cr1-cKO;Ai32^Tg/+ mice. j, Expression of Snx25 and Ngf in BMDMs cultured and sorted from Snx25^Cx3cr1-cKO;Ai32^Tg/+ mice (YFP⁻, n = 3; YFP⁺, n = 3; Snx25, P = 0.027; Ngf, P = 0.009). k, Confocal microscopy of the hind paw skin immunolabeled for YFP and MHC-II in WT mice that received BMT from Snx25^Cx3cr1-cKO;Ai32^Tg/+ mice treated with TAM for 2 weeks. Boxed areas (i–iii) in the upper panel are magnified in the lower panels. Representative of three independent experiments. Scale bar, 100 μm. l, Expression patterns of Snx25 and Ngf in dMacs (n = 3), dMonos (n = 3) and dDCs (n = 3). m, Expression of Snx25 and Ngf in dMacs of WT and Snx25^+/− mice (WT, n = 5; Snx25^+/−, n = 5; Snx25, P = 0.002; Ngf, P = 0.014). n, Expression of Snx25 and Ngf in dMacs of Snx25^fl/fl and Snx25^Cx3cr1-cKO mice (Snx25^fl/fl, n = 3; Snx25^Cx3cr1-cKO, n = 3; Snx25, P = 0.007; Ngf, P = 0.057). o, Expression of Yfp, Snx25 and Ngf mRNA in dMacs of WT mice that received BMT from Snx25^Cx3cr1-cKO;Ai32^Tg/+ mice (YFP⁻, n = 5; YFP⁺, n = 5; Yfp, P = 0.043; Snx25, P = 0.008; Ngf, P = 0.009). p, VF thresholds before and 24 h after injection in WT or Snx25^+/− mice injected with NGF (10 ng μl⁻¹, 10 μl) or PBS (NGF, WT, n = 5, P = 0.017; Snx25^+/−, n = 7, P = 0.014. PBS, WT, n = 4; Snx25^+/−, n = 5). g, gram. Results are represented as mean ± s.e.m. Statistical analyses were performed using two-tailed Student’s t-test (d,e), two-tailed Welch’s t-test (a,b,f,g,j,m–o) or one-way ANOVA (l,p), and significant differences between group means were identified with the Tukey–Kramer test. *P < 0.05, **P < 0.01.

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