Supplementary Figure 1: FAM126A/B are identified as interaction partners of EFR3A/B, and FAM126A is expressed as two splice forms, with the 58 kD splice form the major form in mammalian brain.
From: The leukodystrophy protein FAM126A (hyccin) regulates PtdIns(4)P synthesis at the plasma membrane
(a) HeLa cells stably expressing EFR3A-GFP, EFR3B-GFP, or GFP only were subjected to GFP immunoprecipitation. The purified proteome was digested and analyzed by LC-MS/MS, and peptide and protein identities were determined using MaxQuant. Shown is the sequence coverage of FAM126A (left) and FAM126B (right) from two separate experiments: EFR3A-GFP vs. GFP IP (black bars) and EFR3B-GFP vs. GFP IP (white bars). (b) Top, Domain cartoon of the two splice forms of FAM126A (isoform 1, 58 kD corresponding to NCBI NP_115970.2; isoform 2, 47 kD, corresponding to NCBI XP_005249951.1), with regions common to both splice isoforms in cyan and regions unique to each splice isoform in yellow (isoform 1) and magenta (isoform 2). Bottom, Confirmation of the identity of the 47 kD splice form. Immunoprecipitations using an anti-FAM126A antibody raised against residues 2-308 of human FAM126A or a negative control (rabbit IgG) were performed from lysates of primary human skin fibroblasts and analyzed by SDS-PAGE followed by Coomassie staining. The band at ∼47 kD in the anti-FAM126A sample was excised and analyzed by mass spectrometry. Shown is the list of identified peptides; those colored in magenta are unique to isoform 2 (XP_005249951.1). (c) Immunoblot analysis of FAM126A from lysates from lung of wild-type (WT) and FAM126A knockout (KO) mice. (d) Immunoblot analysis of FAM126A in lysates from gray matter (G) and adjacent white matter (W) samples from human or macaque brain (parietal cortex) and control (C) or patient-derived, FAM126A-deficient (P) primary human skin fibroblasts, demonstrating that isoform 1 is the major form in brain, and isoform 2 is the major form in skin fibroblasts. Arrowheads denote background bands at ∼43 kD in mouse lung (c) and ∼57 kD in fibroblasts (d). Shown are representative immunoblots from three independent experiments.
