Makki K., Froguel P., Wolowczuk I. both and = 6) were intraperitoneally (i.p.) injected with control H2O or 10 nmol of CL, the highly selective agonist of ADRB3 (28, 29), and tissues were harvested 3 h later as described (13, 29). To examine the role of HSL in the ADRB3-regulated SphKs and IL-6 expression, mice (= 7C8) were pretreated with the selective HSL inhibitor BAY or methycelluose as described previously (28,C30). To examine the effect of SphK inhibition on IL-6 expression, mice (= 7C8) were pretreated with SphK inhibitor SKI-2 (20 mg/kg body weight) dissolved in 100 l of dimethylformamide via intraperitoneal injection. Mice injected with dimethylformamide alone were used as a control. One hour later, mice were intraperitoneally injected with 10 nmol of CL or H2O, and tissues were harvested 3 h later. Mice were euthanized, epididymal white adipose tissues (EWAT) pads were collected and processed for real-time PCR analysis and Western blotting analysis. RT-PCR and Real-time PCR Total RNA was isolated from cultured cells and mouse EWAT using the RNeasy kit (Qiagen) and was reversely transcribed with an oligo-dT primer (Promega) by M-MLV reverse transcriptase (Promega) for first strand cDNA synthesis. For real-time PCR quantitation, 50 ng of reversely transcribed cDNAs were amplified with the ABI 7500 system (Applied Biosystems) in the presence of TaqMan DNA polymerase. PCR primer pairs used were as follows: mouse IL-6, 5-AGTGG CTAAG GACCA AGACC-3 (sense) and 5-TCTGA CCACA GTGAG GAATG-3 (antisense); mouse SphK1, 5-TCTAC CTCCC GCCAT AAAA-3 (sense) and 5-CTCCT CCCCA CAACA AAAC-3 (antisense); mouse SphK2, 5-CCAAC AAGTG TCTCC TCCAA A-3 (sense) and 5-CCTCA GGGAT GTCAA AGTTC A-3 (antisense); and mouse GAPDH, 5-CACCT TCGAT GCCGG GGCTG-3 (sense) and 5-GGCCA TGAGG TCCAC CACCC-3 (antisense). The qPCR reaction was performed by using a common PCR Master Blend (Applied Biosystems) relating to manufacturer’s instructions. Relative quantification was determined using the SDS software (Applied Biosystems) based on the following equation: relative quantification = 2?is the threshold cycle to detect fluorescence. values were normalized to the internal GAPDH standard. Sphingolipid Extraction Differentiated 3T3-L1 cells were washed with PBS, changed to phenol-red free simple RPMI 1640, and treated with or without isoproterenol for 3 h. Tradition media were added with 10 l (1 g/ml) of C17-sphingosine internal standard, followed by the addition of equivalent volume of extraction buffer (isopropanol:ethyl acetate = 15:85, v/v). Combination was vortexed for 2 min and then centrifuged for 10 min at 4000 rpm. The top phase was transferred to a new vial, and the aqueous phase was acidified with 100 l of formic acid. The extraction process was repeated with the aqueous phase by adding another equivalent volume of extraction buffer, vortexing for 2 min, and centrifuging for 10 min. The top organic phases from two extractions were combined and speed-vacuum dried. Lipid extracts were reconstituted with 50 l of solvent A (2 mm NH4CO2H, 0.2% formic acid, in 90% H2O Rabbit polyclonal to GLUT1 and 10% methanol) and 50 l of solvent B (95% methanol, 5% H2O, 0.2% formic CCK2R Ligand-Linker Conjugates 1 acid) for LC-MS/MS sphingolipid analysis once we explained (23, 35). LC-MS/MS Quantification For LC-MS/MS analysis, reverse phase HPLC was performed using BDS HYPERSIL C8 columns (100 2.1 mm, 2.4 m, Thermo Scientific) and gradient elution on Waters Alliance 2695 system (Waters Corp.). The mobile phase consisted of methanol, water, and ammonium formate. Solvent A was 2 mm ammonium formate in methanol with 0.2% formic acid. The column was equilibrated with solvent A for 5 min. Samples were injected using the autosampler managed at 10 2 C. The injection volumes were 80 l for each sample. A complete injection of each sample required 7 min, including column equilibration. The circulation rate was 0.3 ml/min. The HPLC eluent was directly launched to QuattroLC mass spectrometer (Micromass-Waters), equipped with an electrospray ion (ESI) resource that was utilized for electrospray ion-MS/MS. The electrospray ion-MS/MS experiments for the quantitation CCK2R Ligand-Linker Conjugates 1 of sphingolipids were performed once we prescribed (23, 33). Briefly, sphingolipid quantitation was carried out in the positive ion mode with ESI needle voltage, 2.8 kV; resource block temp, 120 C; desolvation temp, 350 C; desolvation gas circulation, 540 liters/h; nebulizer gas circulation, 80 liters/h; and the collision gas pressure was 3.2 10?4 pub. Cone voltage and collision energy for each Multiple Reaction Monitoring transition were optimized..M., Lee M. HSL inhibitor BAY or methycelluose as explained previously (28,C30). To examine the effect of SphK inhibition on IL-6 manifestation, mice (= 7C8) were pretreated with SphK inhibitor SKI-2 (20 mg/kg body weight) dissolved in 100 l of dimethylformamide via intraperitoneal injection. Mice injected with dimethylformamide only were used like a control. One hour later on, mice were intraperitoneally injected with 10 nmol of CL or H2O, and cells were harvested 3 h later on. Mice were euthanized, epididymal white adipose cells (EWAT) pads were collected and processed for real-time PCR analysis and Western blotting analysis. RT-PCR and Real-time PCR Total RNA was isolated from cultured cells and mouse EWAT using the RNeasy kit (Qiagen) and was reversely CCK2R Ligand-Linker Conjugates 1 transcribed with an oligo-dT primer (Promega) by M-MLV reverse transcriptase (Promega) for 1st strand cDNA synthesis. For real-time PCR quantitation, 50 ng of reversely transcribed cDNAs were amplified with the ABI 7500 system (Applied Biosystems) in the presence of TaqMan DNA polymerase. PCR primer pairs used were as follows: mouse IL-6, 5-AGTGG CTAAG GACCA AGACC-3 (sense) and 5-TCTGA CCACA GTGAG GAATG-3 (antisense); mouse SphK1, 5-TCTAC CTCCC GCCAT AAAA-3 (sense) and 5-CTCCT CCCCA CAACA AAAC-3 (antisense); mouse SphK2, 5-CCAAC AAGTG TCTCC TCCAA A-3 (sense) and 5-CCTCA GGGAT GTCAA AGTTC A-3 (antisense); and mouse GAPDH, 5-CACCT TCGAT GCCGG GGCTG-3 (sense) and 5-GGCCA TGAGG TCCAC CACCC-3 (antisense). The qPCR reaction was performed by using a common PCR Master Blend (Applied Biosystems) relating to manufacturer’s instructions. Relative quantification was determined using the SDS software (Applied Biosystems) based on the following equation: relative quantification = 2?is the threshold cycle to detect fluorescence. values were normalized to the internal GAPDH standard. Sphingolipid Extraction Differentiated 3T3-L1 cells were washed with PBS, changed to phenol-red free simple RPMI 1640, and treated with or without isoproterenol for 3 h. Tradition media were added with 10 l (1 g/ml) of C17-sphingosine internal standard, followed by the addition of equivalent volume of extraction buffer (isopropanol:ethyl acetate = 15:85, v/v). Combination was vortexed for 2 min and then centrifuged for 10 min at 4000 rpm. The top phase was transferred to a new vial, and the aqueous phase was acidified with 100 l of formic acid. The extraction process was repeated with the aqueous phase by adding another equivalent volume of extraction buffer, vortexing for 2 min, and centrifuging for 10 min. The top organic phases from two extractions were combined and speed-vacuum dried. Lipid extracts were reconstituted with 50 l of solvent A (2 mm NH4CO2H, 0.2% formic acid, in 90% H2O and 10% methanol) and 50 l of solvent B (95% methanol, 5% H2O, 0.2% formic acid) for LC-MS/MS sphingolipid analysis once we explained (23, 35). LC-MS/MS Quantification For LC-MS/MS analysis, reverse phase HPLC was performed using BDS HYPERSIL C8 columns CCK2R Ligand-Linker Conjugates 1 (100 2.1 mm, 2.4 m, Thermo Scientific) and gradient elution on Waters Alliance 2695 system (Waters Corp.). The mobile phase consisted of methanol, water, and ammonium formate. Solvent A was 2 mm ammonium formate in methanol with 0.2% formic acid. The column was equilibrated with solvent A for 5 min. Samples were injected using the autosampler managed at 10 2 C. The injection volumes were 80 l for each sample. A complete injection of each sample required 7 min, including column equilibration. The circulation rate was 0.3 ml/min. The HPLC eluent was directly launched to QuattroLC mass spectrometer (Micromass-Waters), equipped with an electrospray ion (ESI) resource that was utilized for electrospray ion-MS/MS. The electrospray ion-MS/MS experiments for the quantitation of sphingolipids were performed once we prescribed (23, 33). Briefly, sphingolipid quantitation was carried out in the positive ion mode with ESI needle.