2.3. Western Blot

A total of 22 samples from 11 subjects (tumor and adjacent normal tissue) were used for estimation of protein expression of selected enzymes of sphingolipid metabolism. Equal amounts of protein (15 µg) from tumor and normal tissue were separated using SDS–PAGE (Criterion TGX 10% precast gels, Criterion Cell electrophoresis equipment, Bio-Rad, Hercules, CA, USA). The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (TransBlot Turbo semi-dry transfer system, Bio-Rad). After blocking in Tris-buffered saline with 5% nonfat dry milk, the membranes were incubated with an appropriate primary antibody. The following target proteins were quantified using primary antibodies: serine palmitoyltransferase (SPT) (SPTLC2 catalytical subunit, ab23696, Abcam, Cambridge, UK), ceramide synthase 1 (CerS1, ab98062, Abcam), ceramide synthase 2 (CerS2, ab85567, Abcam), ceramide synthase 5 (CerS5, ab73289, Abcam), and cytochrome c oxidase subunit IV (COX IV, #4844, Cell Signaling, Danvers, MA, USA). After incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody, protein bands were visualized with the use of a Bio-Rad ChemiDoc XRS+ system. The protein expression values were normalized to glyceraldehyde 3-phosphate dehydrogenase loading control (GADPH, ab9485, Abcam), measured from parallel runs on separate gel, and expressed as fold changes over control group values. Unless stated otherwise, all the chemicals and equipment used for immunoblotting were purchased from Bio-Rad.