Fig 1: RAMP1/CREB signaling negatively correlates with ECM breakdown in human IVDD NP sample. A) Quantitative analysis of ELISA assay for CGRP concentration in human NP tissue under moderate and severe IVDD grade. B) Representative human lumbar spine MRI images for different IVDD grade ranging from Grade II to Grade V (Moderate: Grade II and Grade III, Severe: Grade IV and Grade V), scale bar: 2 cm. C,D) Quantitative RT-PCR analysis for human NP tissue RAMP1 expression in different IVDD grade ranging from Grade II to Grade V (C) and of human NP tissue from moderate and severe IVDD grade patients (D). E,F) Representative images of WB and quantitative analysis of pCREB expression of human NP tissue from moderate and severe IVDD grade patients. G,H,I) Representative immunohistochemical staining and quantitative analysis of RAMP1 and pCREB expression in the human NP tissue from moderate and severe IVDD grade patients. Scale bar: 100 µm. J) Correlation analysis of relative RAMP1 expression in NP tissue and Pfirrmann grades of patients. K,L,M) Correlation analysis of relative RAMP1 expression in NP tissue and relative CHSY1 (K), ACAN (L), and MMP3 (M) expression. All data are presented as means ± SEM, n = 6 per group (A,H,I), n = 8 per group (F), n = 20 per group (D), n = 40 (J,K,L,M). *P < 0.05. **P < 0.01, statistical significance was determined by two-tailed Student's t-test (A,D,F,H,I). Statistical significance was determined by Pearson correlation analysis (J,K,L,M).
Fig 2: DRG-derived CGRP promotes CHSY1 expression in NP cell via RAMP1/CREB signaling. A) Quantitative RT-PCR analysis of CHSY1 expression from NP cells treated with Vehicle, VIP, CGRP, NPY, and SP at the concentration of 10–7 m, respectively. B,C) Representative images of WB and quantitative analysis of CHSY1 expression for NP cells treated with Vehicle, VIP, CGRP, NPY, and SP. D) Quantitative analysis of CS by DMMB assay for NP cells treated with Vehicle, VIP, CGRP, NPY, and SP. E,F) Representative immunofluorescence staining of CS-56 (green) and quantitative analysis for NP cells treated with Vehicle, VIP, CGRP, NPY, and SP. Scale bar: 50 µm. G) Schematic graph for in vitro co-culture system of DRG sensory neuros and NP cells treated with CGRP neutralizing antibody. H) Quantitative RT-PCR analysis of CHSY1 expression from NP cells in the DRG co-culture system treated with CGRP neutralizing antibody and vehicle. I,J) Representative images of WB and quantitative analysis of CHSY1 expression for NP cells in the DRG co-culture system treated with CGRP neutralizing antibody and vehicle. K) Quantitative analysis of CS by DMMB assay for NP cells in the DRG co-culture system treated with CGRP neutralizing antibody and vehicle. L,M) Representative immunofluorescence staining of CS-56 (green) and quantitative analysis for NP cells in the DRG co-culture system treated with CGRP neutralizing antibody and vehicle. Scale bar: 50 µm. N,O) Representative images of WB and quantitative analysis of pCREB expression for NP cells treated with CGRP for 0–120 min respectively. P,Q) Representative images of WB and quantitative analysis of CHSY1 expression for NP cells treated with CGRP, si-RAMP1, CGRP+si-RAMP1, CREB inhibitor 666-15, and CGRP+666-15, respectively. R) Quantitative analysis of CS by DMMB assay for NP cells treated with CGRP, si-RAMP1, CGRP+si-RAMP1, CREB inhibitor 666-15, and CGRP + 666-15, respectively. All data are presented as means ± SEM, n = 6 per group, *P < 0.05 and **P < 0.01 (A,C,D,F,O,Q,R). Statistical significance was determined by two-tailed Student's t-test. *P < 0.05 and **P < 0.01. Statistical significance was determined by one-way ANOVA (H,J,K,M).
Supplier Page from Abcam for Anti-RAMP1 antibody