Fig 1: Localization and membrane association of SPTLC1 and pathogenic variants.(A) Schematic of SPTLC1 displaying individual protein domains and positions of ALS and HSAN1 pathogenic variants. TMD, transmembrane domain. (B) Confocal images of SPTLC1 localization. WT-SPTLC1FLAG and variants were transiently expressed in COS-7 cells and visualized using an anti-FLAG antibody. ER-mCherry serves as an ER marker. Scale bars: 10 µm. (C and D) Expression of SPTLC1 variants in SPTLC1-KO cells. WT-SPTLC1FLAG and variants were integrated into Flp-In T-REx 293 SPTLC1-KO cells and expressed by addition of tetracycline. Whole-cell lysates were analyzed by SDS-PAGE and immunoblotting with anti-SPTLC1 and anti-SPTLC2 antibodies (C), and SPTLC2 levels were quantified (D). SPTLC2 signals were normalized to the ß-actin signal. Mean ± SD, n = 4 independent replicates, unpaired 2-sided Welch’s t test. **P < 0.01, ***P < 0.001. (E) Analysis of membrane association of SPTLC1 variants. Cell lysates from Flp-In T-Rex 293 control cells and SPTLC1-KO cells expressing WT-SPTLC1FLAG and variants were centrifuged to separate the membrane pellet and cytosolic supernatant. Equal amounts of total (T), pellet (P), and supernatant (S) were analyzed by SDS-PAGE and immunoblotted for VAPB as a membrane protein control and UBB as a cytosolic protein control. See complete unedited blots for C and E in the supplemental material.
Fig 2: Interaction of SPTLC1 variants with ORMDLs.(A) Immunoblot analysis of proteins copurified with SPTLC1 variants. Membrane fractions from Flp-In T-REx 293 control cells and SPTLC1-KO cells expressing WT-SPTLC1FLAG and variants were solubilized by digitonin and subjected to FLAG immunoprecipitation. Input (5%) and eluate (IP: anti-FLAG, 40%) fractions were analyzed by SDS-PAGE and immunoblotting. HSPA5 was used as a negative control. (B and C) Analysis of the SPT complex by blue native PAGE. Membrane fractions from Flp-In T-REx 293 control (CTRL), SPTLC1-KO, and SPTLC2-KO cells (B), or SPTLC1-KO cells expressing WT-SPTLC1FLAG and variants (C) were analyzed by blue native PAGE and immunoblotted with anti-SPTLC1, anti-SPTLC2, and anti-ORMDL antibodies. See complete unedited blots for A–C in the supplemental material. (D) SPT activity in WT HEK293 cells after the siRNA-mediated silencing of ORMDL expression, in the presence or absence of C6-ceramide (C6-Cer). Cells were transfected with either nontargeting scrambled control or isoform-independent ORMDL siRNA. Isotope labeling using D3-15N-L-serine was done 72 hours after transfection. De novo–formed sphingolipids (SLs) were quantified by the incorporation of isotope-labeled D3-15N-L-serine. (E) SPT activity in SPTLC1-deficient HEK293 cells expressing WT SPTLC1 and the SPTLC1-ALS variants L39del and ex2del. Cells were transfected with either scrambled or ORMDL siRNAs. Seventy-two hours after transfection, cells were labeled with D3-15N-L-serine (for 16 hours) and total de novo–formed SLs quantified by LC-MS. (F) SPT activity in WT- or ALS-variant-expressing SPTLC1-KO cells in response to the addition of C6-Cer. Cells were grown with increasing concentrations of C6-Cer. Each data point reflects a single measurement. (G) De novo SL formation in patient-derived primary fibroblasts carrying either the SPTLC1p.L39del or the F40S41del mutation. Control (WT) and mutant fibroblasts were transfected with scrambled or mutant allele–specific siRNA. SL de novo formation was measured in the presence of C6-Cer. The plot shows the total amount of de novo–formed SLs relative to untreated controls. (H and I) Formation of canonical and 1-deoxySL in SPTLC1-KO cells expressing the HSAN1 variants SPTLC1, C133W, S331F, and S331Y. The plot shows total de novo–formed SLs (H) and 1-deoxySLs (I) at the given C6-Cer concentration. SPT activity was measured by the incorporation of D3-15N-L-serine and D4-L-alanine. Mean ± SD, n = 3 independent replicates, 1-way ANOVA with Bonferroni’s adjustment for multiple comparisons. **P < 0.01; ***P < 0.001; ****P < 0.0001.
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