Fig 1: Dose–response and time effects of curcumin on the mRNA expression of SREBP-2, SP-1, and SCAP. (a) mRNA expression at 24 and 48 hr cultivation in different concentrations of curcumin *, p < .05, versus the untreated control. (b) mRNA expression under 25 µM curcumin treatment from 0 ~ 96 hr *, p < .05, versus the untreated control. (c) mRNA expression under 25 µM curcumin treatment from 0 ~ 12h.*, p < .05, versus the untreated control. (d) The comparison of mRNA expression in control group and curcumin group at each time point. *, p < .05, versus the untreated control in each point-in-time. Sketch Map. Normally, INSIG binds to the sterol-sensing domain (SSD) of SCAP (blue transmembrane region in SCAP). As the sterols concentration decreases, the connection between SCAP and INSIG disappears, and the complex of SREBP/SCAP is transported from the endoplasmic reticulum to the Golgi apparatus. S1P and S2P were sequentially involved in cleaving SREBP-2 and releasing the mSREBP-2 into the cell nucleus
Fig 2: Gene and protein expression of components of the PAR-1/SphK/S1P axis. qRT-PCR analysis of the gene expression of (A) PAR-1, (B) S1PR1, (C) SphK1, and (D) SphK2 in astrocytes treated with LPS or thrombin, with or without Dab or PAR-1-inh (LPS only), for 0 h, 1 h, or 6 h. Western blot detection and quantification of the protein expression of PAR-1, S1PR1, SphK1, and SphK2 in astrocytes treated with LPS or thrombin, with or without Dab or PAR-1-inh (LPS only), for (E) 0 h, (F) 1 h, or (G) 6 h. The data are presented as the mean ± SD (n = 3), *p < 0.05.
Fig 3: Immunofluorescence of proteins related to S1P signaling in astrocytes. Astrocytes were treated with LPS or thrombin, with or without Dab or PAR-1-inh (LPS only) for 6 h. Cells were stained for (A) S1P receptor 1 (S1PR1), (B) sphingosine kinase (SphK1), and (C) SphK2 in green and nuclei (DAPI) in blue. Scale bar = 100 µm. Green fluorescence was quantified by measuring the relative mean IOD. The data are presented as the mean ± SD (n = 3), *p < 0.05.
Fig 4: Protein expressions of precursor (p) and nuclear (n) forms of SREBP1 and co-immunoprecipitation assays for interactions of VCP with S1P, RHBDL4, or SREBP1 using liver lysates of WT and A232E/+ mice.A, representative images of Western blot analyses for pSREBP1, pSREBP2, and ß-actin protein levels in the plasma membrane fraction of the liver tissue. B and C, ratio of signal intensities of pSREBP1 (B) and pSREBP2 (C) to ß-actin in Western blot analyses. D, representative images of Western blot analyses for nSREBP1, nSREBP2, ChREBP, and TBP protein levels in the nuclear fraction of the liver tissue. E–G, ratio of signal intensities of nSREBP1 (E), nSREBP2 (F), and ChREBP (G) to TBP in Western blot analyses. H, immunoblots for S1P and VCP using proteins immunoprecipitated with anti-S1P antibody. I, immunoblots for RHBDL4 and VCP using proteins immunoprecipitated with anti-RHBDL4 antibody. J, immunoblots for SREBP1 and VCP using proteins immunoprecipitated with anti-SREBP1 antibody. Liver lysates of mice fed HFD were used for co-immunoprecipitation assays. Input indicates liver lysates without immunoprecipitation. IgG indicates control proteins immunoprecipitated with isotype antibody. Spliced-together lanes were run on the same gel but were noncontiguous. B, C, E–G, values are means ± SEM (SD, n = 5 per group; HFD, n = 12–13 per group). Fold change is relative to WT mice fed SD. *p < 0.05, **p < 0.01, ***p < 0.005 as determined by 2-tailed Student’s t test for comparing two groups. ChREBP, carbohydrate-responsive element–binding protein; HFD, high-fat diet; SD, standard diet; S1P, site-1 protease; SREBP1, sterol regulatory element–binding protein 1; TBP, TATA box-binding protein; TG, triglyceride; VCP, valosin-containing protein.
Fig 5: The expression of S1P/S2P mRNA and protein under 25 µM curcumin treatment. (a) The change of mRNA expression. *, p < .05, versus the untreated control (first column). (b) The Western blot and the gray value analysis results. ß-actin was used as an invariant control for equal loading. *, p < .05, versus the untreated control (first column)
Supplier Page from Abcam for Anti-S1P antibody