HC was purchased from Yamakawa (Tokyo, Japan). HC extracted from horse spinal cord is a mixture of lipids containing GalCer(72~82%) as the main component along with sphingomyelin, phosphatidylethanolamine, lecithin (combined 8~25%) and cholesterol (0.1~0.6%). The GalCer of HC contains more than 11 types of molecular species. The structures with the highest content in HC are d18:14E-C24:0GalCer and d18:14E-C24h:0 GalCer.
Ceramide standards, Cer[NS/NDS], Cer[NP] and Cer[AS], used for quantitative determination, were purchased from Takasago International Corp. (Tokyo, Japan), Evonik Industries (Essen, Germany) and Matreya (State College, PA, USA). RHEEs (LabCyte EPIMODEL 6D) and assay medium were purchased from Japan Tissue Engineering (Aichi, Japan). NHKs and assay medium (Humedia-KG2) were purchased from Kurabo (Tokyo, Japan). PBS (-) was purchased from Fujifilm Wako Pure Chemical Corp. (Osaka, Japan), 0.05% Trypsin-EDTA was purchased from Gibco (BRL, Palo Alto, CA, USA) and fetal bovine serum (FBS) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Isogen was purchased from Nippon Genes (Tokyo, Japan). The One-step Syber Prime Script PLUS RT-PCR kit was purchased from Takara Bio (Shiga, Japan). PCR primers were purchased from Hokkaido System Science (Hokkaido, Japan). IGEPAL™ CA-630 and cOmpl™TM) Mini Protease Inhibitor Cocktail Tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA). The DC Protein Assay Kit, Laemmli sample buffer and Clarity Western ECL Substrate were from Bio-Rad Laboratories (Hercules, CA, USA). Can Get Signal solutions 1 and 2 were purchased from Toyobo (Osaka, Japan). Antibodies to ELOVL4 (DF4037) were obtained from Affinity Biosciences (Cincinnati, OH, USA). Antibodies to CERS3 (Anti-LASS3: ab169259), ASM (ab83354) and INV (ab53112) were obtained from Abcam (Cambridge, UK) and TGM3(NBP1-86950) was from Novus Biologicals (Littleton, CO, USA). Antibodies to SMS1 (bs-4216R) and GCS (bs-8593R) were obtained from Bioss (Beijing, China). Antibodies to GBA (G4171) and beta-Actin (A1978) and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG and HRP conjugated goat anti-rabbit IgG were obtained from Sigma-Aldrich (St. Louis, MA, USA). Antibodies to TGM1 (12912-3-AP) were obtained from Proteintech Group (Rosemont, IL, USA). Agonists and antagonists to PPARs, WY14643/GW501516, GW6471/GSK0660 and Rosiglitazone/GW9662 were obtained from Adipogen Life Sciences (San Diego, CA, USA), Cayman Chemical (Ann Arbor, MI, USA) and Fujifilm Wako Pure Chemical Corp. (Osaka, Japan), respectively. TLC plates for HPTLC were obtained from Merck Millipore (Darmstadt, Germany). All other reagents were obtained commercially and used without further purification.
Ceramide standards, Cer[NS/NDS], Cer[NP] and Cer[AS], used for quantitative determination, were purchased from Takasago International Corp. (Tokyo, Japan), Evonik Industries (Essen, Germany) and Matreya (State College, PA, USA). RHEEs (LabCyte EPIMODEL 6D) and assay medium were purchased from Japan Tissue Engineering (Aichi, Japan). NHKs and assay medium (Humedia-KG2) were purchased from Kurabo (Tokyo, Japan). PBS (-) was purchased from Fujifilm Wako Pure Chemical Corp. (Osaka, Japan), 0.05% Trypsin-EDTA was purchased from Gibco (BRL, Palo Alto, CA, USA) and fetal bovine serum (FBS) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Isogen was purchased from Nippon Genes (Tokyo, Japan). The One-step Syber Prime Script PLUS RT-PCR kit was purchased from Takara Bio (Shiga, Japan). PCR primers were purchased from Hokkaido System Science (Hokkaido, Japan). IGEPAL™ CA-630 and cOmpl™TM) Mini Protease Inhibitor Cocktail Tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA). The DC Protein Assay Kit, Laemmli sample buffer and Clarity Western ECL Substrate were from Bio-Rad Laboratories (Hercules, CA, USA). Can Get Signal solutions 1 and 2 were purchased from Toyobo (Osaka, Japan). Antibodies to ELOVL4 (DF4037) were obtained from Affinity Biosciences (Cincinnati, OH, USA). Antibodies to CERS3 (Anti-LASS3: ab169259), ASM (ab83354) and INV (ab53112) were obtained from Abcam (Cambridge, UK) and TGM3(NBP1-86950) was from Novus Biologicals (Littleton, CO, USA). Antibodies to SMS1 (bs-4216R) and GCS (bs-8593R) were obtained from Bioss (Beijing, China). Antibodies to GBA (G4171) and beta-Actin (A1978) and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG and HRP conjugated goat anti-rabbit IgG were obtained from Sigma-Aldrich (St. Louis, MA, USA). Antibodies to TGM1 (12912-3-AP) were obtained from Proteintech Group (Rosemont, IL, USA). Agonists and antagonists to PPARs, WY14643/GW501516, GW6471/GSK0660 and Rosiglitazone/GW9662 were obtained from Adipogen Life Sciences (San Diego, CA, USA), Cayman Chemical (Ann Arbor, MI, USA) and Fujifilm Wako Pure Chemical Corp. (Osaka, Japan), respectively. TLC plates for HPTLC were obtained from Merck Millipore (Darmstadt, Germany). All other reagents were obtained commercially and used without further purification.
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