Fig 1: CB1R-BiP complexes reside on GABAergic terminals of the mouse striatum and cortex. A, PLA experiments were conducted on striatal and cortical sections from 3- to 4-month-old mice of different genotypes. Representative low-magnification image and selected regions for analysis are shown. Image credit: Allen Institute. In the rest of the panels, CB1R-BiP complexes are shown as red dots, and nuclei are colored in blue by DAPI staining. B, Representative images of striatal sections from CB1R-floxed, CB1R-KO, GABA-CB1R-KO, and Glu-CB1R-KO mice. C, Representative images of striatal sections from Stop-CB1R, CB1R-RS, GABA-CB1R-RS, and Glu-CB1R-RS mice. D, Quantification of the number of cells containing one or more dots expressed as the percentage of the total number of cells (DAPI-stained nuclei) in striatal sections. **p < 0.01 from the corresponding CB1R-floxed group or the corresponding CB1R-RS group by one-way ANOVA with Tukey's multiple comparisons test (n = 6 or 7 fields from 3 different animals per group). E, Representative images of cortical sections from CB1R-floxed, CB1R-KO, GABA-CB1R-KO, and Glu-CB1R-KO mice. F, Representative images of cortical sections from Stop-CB1R, CB1R-RS, GABA-CB1R-RS, and Glu-CB1R-RS mice. G, Quantification of the number of cells containing one or more dots expressed as the percentage of the total number of cells (DAPI-stained nuclei) in cortical sections. **p < 0.01 from the corresponding CB1R-floxed group or the corresponding CB1R-RS group by one-way ANOVA with Tukey's multiple comparisons test (n = 6 or 7 fields from 3 different animals per group).
Fig 2: Changes in ER stress markers after rhizotomy. (A) Quantitative bar graphs and (B) immunoblots and of Sig-1R, BiP, phosphorylated IREα/IRE ratio, XBP-1s/XBP-1unspliced ratio and CHOP proteins in control (CTL) and in injured mice with or without treatment with PRE-084 and BD1063 at 7 and 14 dpi. n = 3–4 per group and time point. Data are mean ± SEM and analyzed with One-way ANOVA and Dunnett’s multiple comparisons test. ### p < 0.001 vs. CTL mice; ** p < 0.005, * p < 0.05 vs. saline 14 dpi. (C) Microphotographs of XBP-1 labeling in the ventral horn of spinal cord confirms the upregulation of this ER stress marker in MNs at 7 dpi. Scale bar 50 μm. (D) Immunoblots and (E) bar graphs of BiP, phosphorylated IREα/IRE ratio and XBP-1s/XBP-1unspliced ratio, in WT and Sig-1R KO mice groups at 14 dpi. n = 3–5 per group. One-way ANOVA and Dunnett’s post-hoc test: # p < 0.05 vs. CTL mice.
Fig 3: Insulin-mediated enhancements in STAT3 signaling were mediated through GRP78 in SH-SY5Y-ObRb cells.SH-SY5Y-ObRb cells were transfected with siRNA (5 nM) for 72 h. Seventy-two hours after the transfection, medium was changed to serum-free medium for another 20 h. Cells were treated with insulin (300 nM) for 4 h followed by leptin (0.03 μg/ml, 15 min). Phospho-STAT3 (Tyr705), STAT3, GRP78, and GAPDH levels were analyzed by Western blotting. (A) Leptin-induced STAT3 phosphorylation in insulin-treated cells was ameliorated in GRP78-knocked down cells. (B) A densitometric analysis of phospho-STAT3 (Tyr705) and GRP78 was performed using image analysis software. Data are expressed as the mean ± S.E. of 4 independent experiments (n = 4). **P < 0.01.
Fig 4: Expression of BiP and CB1R mRNA in the mouse brain. A, B, Representative autoradiographic images of coronal sections from adult mouse brain showing the mRNA hybridization pattern of BiP (A) and CB1R (B). CA, Cornu ammonis; DG, dentate gyrus; Str, striatum; Cx, cortex; Cb, cerebellum. C, Distribution of CB1R mRNA in the mouse hippocampus. Ci, Representative dark field image from a section hybridized with 33P-labeled oligonucleotide probes for CB1R mRNA. A positive signal is evident as clusters/accumulation of bright silver grains. Note the moderate signal on the pyramidal cell layer of CA and the very intense signal on scattered cells in the various hippocampal layers. Cii, Ciii, Colocalization of CB1R mRNA and BiP mRNA in cells of the stratum radiatum and stratum lacunosum moleculare of CA. Pyr, Pyramidal cell layer of CA. Civ, Cv, Colocalization of CB1R mRNA and BiP mRNA in cells of the polymorphic layer (Pl). Gr, Granular cell layer. Sections were hybridized with 33P-labeled probes for CB1R mRNA (signal visualized as clusters of bright silver grains in dark field images) and with digoxigenin-labeled probes for BiP mRNA (signal visualized as dark precipitate in bright field images). Arrows point to some double-labeled cells. D, Quantification of CB1R mRNA-positive cells that coexpress BiP mRNA (n = 4 mice per group).
Fig 5: Activation of UPR pathway in VCT cells under LPV exposition. Cytotrophoblast cells isolated from human term placentas were cultured for 72 h before incubation with LPV 10 µM or DMSO for 6 h. (A) mRNA expression of sXBP1 and GRP78 were measured by RT-qPCR. The results are expressed as mean +/- SD of n = 3 independent experiments. Two-tailed paired no parametric t-test were realized. (B) GRP78 protein expression was evaluated by immunoblotting with anti-GRP78 antibody. Actin protein determined with anti-actin antibody was used as loading control. The results are expressed as mean +/- SD of n = 6 independent experiments. Two-tailed paired no parametric t-test were realized.
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