Levels of Sph 1-P and dhSph 1-P in various tissues.

Levels of Sph 1-P and dhSph 1-P in various tissues.</p

Levels of total Cer, dhC16-Cer, Sph and dhSph in various tissues.

Levels of total Cer, dhC16-Cer, Sph and dhSph in various tissues.</p

Predominant Cn-Cer species in eight tissues.

Results are presented as pmol Cn-Cer/mg protein with means ± st dev. of 6x replicates. A. C18-Cer; B.C18:1-Cer; C. C20-Cer; D. C20:1-Cer; E. C26-Cer; F. C26:1-Cer.</p

Levels of C18, C18:1-Cer; C20, C20:1-Cer; C26, C26:1-Cer in various tissues.

Levels of C18, C18:1-Cer; C20, C20:1-Cer; C26, C26:1-Cer in various tissues.</p

Cn-Cer’s profile in various tissues.

Results are presented as pmol Cn-Cer/mg protein with means ± st dev. of 6x replicates. A. Brain; B. Heart; C. Kidney; D. Lung; E. Bladder; F. Spleen; G. Liver; H. Blood plasma; I. Tumor xenograft.</p

Levels of total Cer, Sph, dhC16-Cer, and dhSph present in various tissues.

Tissues were homogenized in lysis buffer containing protease-inhibitor cocktail before centrifugation to get protein. Then equal amounts of protein (800ug) were provided for analysis of SL. Results are presented as pmol SL/mg protein with means ± st dev. of 6x replicates. A.Total Cer, * pvs brain), ** pvs liver); B.dhC16-Cer, * pvs tumor), ** pvs liver); C. Sph, * pvs kidney), ** pvs heart); D. dhSph, * pvs brain), ** pvs bladder).</p

Levels of total phosphate in various tissues.

Results are presented as nmol/mg protein with means ± st dev. of 6x replicates. * pvs brain), ** pvs bladder).</p

Major Cn-Cer species (>10% of total Cer) in various tissues.

Major Cn-Cer species (>10% of total Cer) in various tissues.</p

SDS-PAGE of purified RsASML protein.

<p>(a) SDS-PAGE of RsASML protein purification stained with coomassie blue. The arrow indicates the final protein purity used in biochemical assays. Abbreviations: SN: supernatant, FT: flow-through from Ni-Column, Elut: Elution from Ni-Column, Cut: Elution fractions incubated with ULP-1, a SUMO specific-protease, SEC: Size-exclusion chromatography. (b) SDS-PAGE of RsASML protein ran under non-reducing (−βME) and reducing (+βME) conditions induces a band shift, consistent with RsASML containing intra-molecular disulfide bonds.</p

Michaelis-Menten kinetics of RsASML vs. different pNP-based substrates.

<p>Concentration dependence of RsASML activity towards (a) pNPP, (b) pNPPC, (c) pNP-TMP. All reactions were carried out in 10 mM HEPES, pH 8.0, 1 mM NiCl₂, with 100 nM RsASML protein.</p