Figure 2Mean ± SEM CB1 mRNA levels in cultured hippocampal neurons from WT and ASM-KO mice (****P < 0.0001, n = 5 independent cultures, Student's t-test).Western blot against CB1 and GAPDH (used as loading control). Images belong to the same blot but to non-consecutive lanes as indicated by the boxes. Graph showing mean ± SEM CB1 protein levels in cultured neurons from WT and ASM-KO mice (***P= 0.0009, n = 5 independent cultures, Student's t-test).Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from WT and ASM-KO mice. TOPRO stains cell nuclei. Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units (left) or the Mander´s coefficient for the colocalization of CB1 with Lamp1 (right) (****P < 0.0001, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in Purkinje cells of the cerebellum from WT and ASM-KO mice. Graph shows mean ± SEM Mander´s coefficient indicative of the degree of colocalization of CB1 and Lamp1 (*P= 0.0213, n = 4 mice per group, Student's t-test). Scale bar, 5 μm.Graphs show mean ± SEM SM levels, expressed as percentage of WT values, in extracts containing the same amount of protein of cultured neurons from WT and ASM-KO mice (E)Graphs show mean ± SEM SM levels, expressed as percentage of WT values, in extracts containing the same amount of protein of cultured neurons from WT mice incubated or not with 40μΜ SM (F).Mean ± SEM CB1 mRNA levels in cultured neurons from WT mice incubated or not with 40μM SM (**P = 0.0021, n = 5 independent cultures, Student's t-test).Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice incubated or not with 40μΜ SM (n = 2 independent cultures, Student's t-test)Immunofluorescence images against CB1 and Lamp1 in cultured neurons from WT mice incubated or not with 40 μΜ SM. Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units (left) or the Mander´s coefficient for the colocalization of CB1 with Lamp1 (right) (***P <0.0001, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice treated with vehicle (veh), with 40μM SM (SM), with 0,1μM Bafilomycin (BafA1) or with 40μM SM and 0,1μM Bafilomyecin (SM+BafA1). Graph shows mean ± SEM CB1 levels normalized to GAPDH in arbitrary units (n = 2 independent cultures, one - way ANOVA).Mean ± SEM SM levels in cultured neurons from ASM-KO mice incubated or not with exogenous SMase (*P = 0.0157, n = 3 independent cultures, Student's t-test).Mean ± SEM CB1 mRNA levels in cultured neurons from ASM-KO mice incubated or not with exogenous SMase (**P= 0.0015, n = 5 independent cultures, Student's t-test).Western blot against CB1 and GAPDH (used as loading control). Images belong to the same blot but to non-consecutive lanes as indicated by the boxes. Graph showing mean ± SEM CB1 protein levels normalized to GAPDH in extracts from cultured neurons from ASM-KO mice incubated or not with exogenous SMase (n = 5 independent cultures, Student's t-test).Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from ASM-KO mice incubated or not with exogenous SMase. DAPI stains cell nuclei. Graph shows mean ± SEM Mander´s coefficient for the colocalization of CB1 with Lamp1 (*P = 0.0189, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.