Fig 1: (a) RT-PCR analysis of Ucn (upper panel) and GAPDH (lower panel) expression in control (untreated) C-20/A4 chondrocytes and cells treated with 1 mM SNAP and 70 pg/ml TNF-a. Ucn and GAPDH amplicon sizes are indicated. (b) Ratio of Ucn/GAPDH expression in control (untreated) C-20/A4 chondrocytes and cells treated with 1 mM SNAP and 70 pg/ml TNF-a as determined by densitometric analysis of PCR products shown in a. (c) RT-PCR analysis of CRFR subtype expression in C-20/A4 chondrocytes. Lanes 1 and 2=CRFR1 product, lane 3=CRFR1-negative control, lanes 5 and 6=CRFR2 product, lane 7=CRFR2-negative control. CRFR amplicon sizes are indicated. (d) RT-PCR analysis of CRFR2 splice variant expression in C-20/A4 chondrocytes. Lanes 1 and 2=CRFR2a PCR – no product, lane 3=CRFR2a-negative control, lanes 5 and 6=CRFR2ß PCR product, lane 7=CRFR2ß-negative control, lanes 9 and 10=CRFR2? PCR – no product, lane 11=CRFR2?-negative control. CRFRß amplicon size is indicated
Fig 2: Three-weeks of SF selectively increases CRH system activity in the brain. (A): three-weeks of SF increases the levels of CRH, CRHR1, and CRHR2 levels in the HP (n = 6, * p < 0.05); (B,C): three-weeks of SF has no effects on CRH system in the striatum and PFc.
Fig 3: The effects of short-term SF on CRF signaling in extrahypothalamic regions. (A) CRFR2 but not CRFR1 significantly increased its levels in the striatum of SF-experienced mouse brains compared to sham controls (* p < 0.05, n = 5). (B) CRFR1 and CRFR2 did not significantly increase their levels in the hippocampus of SF-experienced mouse brains compared to sham controls (p > 0.05, n = 5). (C) CRFR1 and CRFR2 did not significantly increase their levels in the frontal cortex of SF-experienced mouse brains compared to sham controls. (D) CRF did not significantly increased its mRNA levels in the striatum, hippocampus, or frontal cortex of SF-experienced mouse brains compared to sham controls. For all Western blots, ß-actin were served as a protein load control. For all quantitative polymerase chain reactions (qRT-PCR), GAPDH were served as an internal control for quantification. Data were expressed as means ± SEM and were analyzed using Student’s t-tests (* p < 0.05 versus sham). (Original Western Blot Figure see Supplementary Figure S5).
Fig 4: (a) Western-blot analysis of CRFR expression. CRFR1 and CRFR2 protein levels were determined using rabbit anti-CRFR1 and rabbit anti-CRFR2 monoclonal antibodies with a mouse anti-a-tubulin monoclonal antibody used as a loading control. Bands with sizes corresponding to 50 kDa (CRFR1), 47 kDa (CRFR2) and 55 kDa (a-tubulin) were detected. (b) Observed bands were quantified by densitometry and CRFR expression normalized to that of a-tubulin. Data are presented as mean±S.E.M. of three independent experiments
Fig 5: The effects of cocaine on CRF signaling in the striatum of both sexes. (A) Cocaine significantly increased CRF levels in male but not female striatum (n = 5, * p < 0.05). (B) Cocaine had no effects on CRFR1 in the striatum of either sex. (C) Cocaine had no effects on CRFR2 in the striatum of either sex.
Supplier Page from MilliporeSigma for Anti-CRHR1 antibody produced in rabbit