C6 nbd ceramide

C6-NBD-ceramide, NVP-231, LY-294002, KICqStart^®SYBR^®Green qPCR ReadyMix™ (SYBRgreen), hCerK MISSION^R shRNA glycerol stocks, and the anti-β-actin (clone AC-15) antibody were from Sigma-Aldrich Chemie GmbH (Buchs, Switzerland). U-0126 was from InvivoGen (Nunningen, Switzerland). Y-27632 was from Tocris (Zug, Switzerland). Fluorescently-labeled Odyssee IRdye 800CW secondary antibodies were from LI-COR Biosciences (Bad Homburg, Germany). Primers for qPCR were from Eurofins Genomics Germany GmbH (Ebersberg, Germany). The First Strand DNA Synthesis Kit was from ThermoFisher Scientific (Zug, Switzerland) and RNAsolv^® from VWR International AG (Dietikon, Switzerland). Antibodies against phospho-Ser⁴⁷³-Akt and total Akt were from Cell Signaling/BioConcept (Allschwil, Switzerland).

Macrophages were pretreated with 0, 0.33, 1.1, 3.3, 11, 33, 100 μM YE2. After 2 h, 2 μg/ml C6-NBD-ceramide (Sigma–Aldrich) was added. The supernatant was collected, and a solution of chloroform:methanol (volume ratio 2:1) was added for lipid extraction 2 h later. Then, the solution was centrifuged at 10,000 rpm for 10 min, and the lower organic phase was collected, dried with N₂, and reconstituted in 30 μL chloroform. Finally, the product NBD-SM was separated by TLC using CHCL3:CH3OH:NH3·H2O, 16:4:1 (v/v/v) as the developing solvent. The bands were observed through UV light detection (Ding and Jiang, 2014 (link)).