Panel A shows a sequence alignment of the M Y C T 1 protein across various vertebrate species, with amino acid conservation color-coded. Percentages indicate sequence identity and homology compared to the human sequence. Panel B presents a Western blot analysis of cells transduced with A d-G F P or A d-M Y C T 1, showing the expression of M Y C T 1 and other proteins. Panel C outlines the workflow for mass spectrometry experiments used to identify M Y C T 1 interactors. Panel D features a Venn diagram that illustrates the selection process for M Y C T 1 interactors. Panel E displays the interaction between M Y C T 1 and I F I T M 2 slash 3 analyzed by proximity ligation assay (P L A) in endothelial cells, with staining for P L A dots, V E-cadherin, and D N A. Panel F quantifies the number of P L A dots per cell under different conditions, showing a significant reduction in M Y C T 1 knockdown cells. Panel G includes positive and negative controls for the P L A signal in human brain sections, with staining for V E-cadherin or P E C A M 1 and D N A. The graphs and images collectively demonstrate the conservation, expression, and interaction of M Y C T 1 across different experimental setups.
MYCT1 is a transmembrane phosphoglycoprotein that interacts with IFITM2/3, related to Fig. 5. (A) The MYCT1 protein is highly conserved across vertebrates. An alignment of human MYCT1 protein sequence with those of the indicated species, amino acid conservation is color-coded as indicated in the legend below. Percentages next to species indicate amino acid sequence identity (left) and homology (right) in comparison with human sequence. (B) Short MYCT1 isoform is predominant in ECs. Cells were transduced with Ad-GFP (control) or Ad-MYCT1 (187-aa isoform with C-terminal V5 tag) adenoviruses. Western blot analysis for the indicated proteins. (C) Workflow for mass spectrometry experiments. MYCT1-negative SW480 colon cancer cells were used to exclude nonspecific interactors pulled down by MYCT1 IgG. n = 2 independent experiments. (D) Venn diagram showing how the short list of MYCT1 interactors was selected. (E) Interaction between MYCT1 and IFITM2/3 was analyzed by PLA in ECs. siRNA-mediated knockdown of either protein confirmed specificity of PLA signal. Staining of ECs for PLA dots (gray), VE-cadherin (magenta), and DNA (blue). Scale bar, 50 µm. (F) Quantification of the number of PLA dots per cell in control, MYCT1KD, and IFITM2/3KD cells. n = 3 independent experiments; 500–1,500 cells were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Dunnett’s multiple comparisons, P = 0.036 (*) for MYCT1 knockdown effect. (G) Positive control (VE-cadherin::β-catenin) and negative control (MYCT1 antibody alone) for PLA signal in human brain sections. PLA dots (gray) and staining of ECs for VE-cadherin or Pecam1 (magenta) and DNA (blue). Arrowhead, colocalization of PLA dots with vascular marker. Scale bar, 20 µm. Source data are available for this figure: SourceData FS4.