(10?ml) each of buffers B, C (buffer A containing 0

(10?ml) each of buffers B, C (buffer A containing 0.2% digitonin) and D (buffer A containing 0.2% Triton X-100), and finally with 8 vol. following. First, homogenates from COS-7 cells transfected with mouse cDNA exhibited C-4-hydroxylase activity in an assay with mRNA content was regulated in a tissue-specific manner and was high in the mouse small intestine. Thirdly, hybridization detected mRNA in mouse crypt cells. Fourthly, immunohistochemistry using an anti-(Des2 peptide) antibody stained mouse crypt cells and the adjacent epithelial cells [17]. Glycolipids with 4-hydroxysphinganine are found only in the intestine, kidney KRas G12C inhibitor 1 and skin, with the highest content observed in the small intestine [6C13,18]. We measured dihydroceramide:sphinganine C-4-hydroxylase activity in the SERPINE1 homogenates or microsomal fractions of mouse small intestine and kidney. We were able to detect the C-4-hydroxylase activity in the kidney but not in the small intestine owing to very high ceramidase activity. We chose to express Des2 as a recombinant protein and to reconstitute the C-4-hydroxylase activity using purified components. We have discussed previously that, as a preliminary result, the addition of cytochrome using purified FLAG-tagged Des2, cytochrome cDNA, as described previously [17], was subcloned into the pFLAG-MAC vector (Sigma), adding a nucleotide sequence encoding the FLAG epitope (DYKDDDDDK) to the 5-end of the cDNA. The region coding for FLAGCDes2 (1?kb) was subcloned into the pFastBac1 vector and the recombinant plasmid was used to transform DH10Bac? competent supernatant, detergent solubilized fractions and fractions collected before and after ultracentrifugation) or purified FLAGCDes2 in a total volume of 50?l. The reactions were initiated by the addition of 100?mM NADH dissolved in 10?mM Tris/HCl, pH?7.5, and 1?mM EDTA at a final concentration of 5?mM, and were allowed to proceed for 2?h at 37?C. Reactions were terminated by the addition of 150?l of 50?mM Tris/HCl, pH?7.5, and 570?l of chloroform/methanol (2:1, v/v). Lipids were recovered from the lower organic phase. The dried lipids collected KRas G12C inhibitor 1 at the bottoms of conical tubes were dissolved in 20?l of chloroform/methanol (1:1, v/v). All the lipids were applied to borate-impregnated HPTLC plates using a microsyringe. Chloroform/methanol/water (60:20:2, by vol.) was used as the developing solvent. The lipids produced by the enzyme reaction were visualized and quantified by autoradiography with a Fuji Bas 2500 bio-imaging analyser. To identify spots by autoradiography, unlabelled for 10?min. The supernatant obtained was then solubilized with 1% (v/v) of each of the following detergents: digitonin, deoxycholate, octylglucoside or Triton X-100 at a protein concentration of 2?mg/ml for 1?h on ice and centrifuged at 50000?rev./min in a Beckman TLA110 rotor for 1?h at 4?C. Samples (volume dependent KRas G12C inhibitor 1 on experiment) of the supernatants and pellets were analysed by Western blotting with the anti-FLAG M2 monoclonal antibody (Sigma). To purify FLAGCDes2, the supernatant obtained from 6108?cells by centrifugation at 600?for 10?min was solubilized with 1% digitonin for 1?h on ice and centrifuged at 50000?rev./min in a Beckman TLA110 rotor for 1?h at 4?C. The resulting supernatant was adjusted to 0.2?M NaCl and 0.2% digitonin in buffer A, and applied to an anti-FLAG M2 antibody-conjugated agarose column (2?ml; Sigma) pre-equilibrated with buffer B (buffer A containing 0.2?M NaCl and 0.2% digitonin). The column was washed with 5 vol. (10?ml) each of buffers B, C (buffer A KRas G12C inhibitor 1 containing 0.2% digitonin) and D (buffer A containing 0.2% Triton X-100), and finally with 8 vol. of buffer C. The bound FLAGCDes2 was eluted with 5 vol. of buffer E (buffer C containing 100?g/ml FLAG peptide). The fractions containing active enzyme were collected, concentrated using an Ultra-4 centrifugal filter (Millipore) and stored at ?80?C in a 1?M sucrose solution. The purified FLAGCDes2 was analysed by SDS/PAGE and Western blotting (see below). All procedures were performed at 4?C unless stated otherwise. Protein concentrations were determined using the Bradford reagent or the BCA (bicinchoninic acid) method with BSA as the reference. The amount of purified FLAGCDes2 was estimated by densitometry of silver-stained SDS/PAGE gels with FLAG-tagged bacterial alkaline phosphatase (Sigma) as a reference. SDS/PAGE,.