ifitm3 human reverse  (Eurofins)

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: MYCT1 is a transmembrane phosphoglycoprotein that interacts with IFITM2/3. (A) Endogenous MYCT1 is located at cell–cell junctions (arrow) and in puncta (arrowhead). Staining of human primary ECs for MYCT1 (black), VE-cadherin (magenta), and DNA (blue). Scale bar, 10 µm. (B) MYCT1 is a membrane protein. Western blot analysis of various EC fractions for MYCT1, GAPDH, PECAM1, H3K27ac, and vimentin proteins. Cy, cytoplasm; Mb, membrane; Nu, nucleus; Ck, cytoskeleton. (C) MYCT1 is glycosylated. Western blot analysis of MYCT1 protein electrophoretic mobility in control and PNGase-F–treated lysates. (D) Schematic model of MYCT1 structure and domains with phosphorylation sites, identified by mass spectrometry. MYCT1 phosphorylation sites are highly conserved as indicated by the color scale. Asterisks indicate sites also described at https://www.phosphosite.org/ . (E) Top five proteins interacting with MYCT1 as identified by mass spectrometry, among which IFITM2 and IFITM3. ECs were transduced with recombinant adenoviruses to transiently overexpress MYCT1 or GFP, as a control. Cell lysates were collected 48 h after transduction, immunoprecipitated using MYCT1 antibody or a control IgG, and analyzed by mass spectrometry. Proteins interacting with both endogenous and overexpressed MYCT1 were selected and ranked by normalized spectral abundance factor (NSAF) from two independent mass spectrometry (MS) experiments are shown (31 proteins); the top five proteins are highlighted in magenta. (F) GO terms of the cellular component and biological process overrepresented in the MYCT1 interactome. Fisher’s exact test with adjustment for false discovery rate (FDR). (G) Validation of IFITM2/3 and MYCT1 interaction by co-IP. EC lysates from confluent ECs were immunoprecipitated (IP) with MYCT1 or control IgG and blotted for IFITM2/3. H, IgG heavy chain; L, IgG light chain. (H) IFITM2/3 are constitutively expressed in ECs in vitro and in vivo . Staining of human primary ECs (upper panels), human brain and WAT sections (lower panels) for MYCT1 (gray), IFITM2/3 (green), VE-cadherin (magenta), and DNA (blue). Scale bar, 20 µm (brain) and 50 µm (adipose tissue). (I) MYCT1 and IFITM2/3 interact in brain ECs. Proximity ligation assay (PLA) in human brain sections. Detection of PLA dots (gray) in ECs and staining for VE-cadherin (magenta) and DNA (blue). Arrowheads, colocalization of MYCT1::IFITM2/3 PLA dots and VE-cadherin staining. Scale bar, 20 µm. See also . Source data are available for this figure: .

Article Snippet: IFITM3 (human) reverse , Eurofins , 5′-CCA GCA CAG CCA CCT CG-3′.

Techniques: Staining, Membrane, Western Blot, Control, Phospho-proteomics, Mass Spectrometry, Transduction, Recombinant, Immunoprecipitation, Biomarker Discovery, Co-Immunoprecipitation Assay, In Vitro, In Vivo, Proximity Ligation Assay

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: MYCT1 restricts endothelial endocytosis and IFITM2/3-dependent mTORC1 activation, related to Figs. 6 and 7. (A) IFITM2/3 antibody and siRNA validation for identification of endogenous human IFITM2/3 proteins. IFITM2/3 knockdown reduces MYCT1 protein levels. Staining of ECs for MYCT1 (gray), IFITM2/3 (green), and DNA (blue). Scale bar, 20 µm. (B) Quantification of MYCT1 protein levels in control and IFITM2/3 KD cells. n = 4 independent experiments; mean ± SD; Welch’s t test, P = 0.0014 (*). (C and D) MYCT1 knockdown does not affect IFITM2 (C) nor IFITM3 (D) mRNA levels in ECs. n = 3 independent experiments; mean ± SD; Welch’s t test, P > 0.05. (E) MYCT1 knockdown does not impact RAB7 + late endosomes nor LAMP1 + endolysosomes. Staining of ECs for RAB7 (gray), LAMP1 (green), VE-cadherin (magenta), and DNA (blue). Scale bar, 20 µm. (F and G) Quantification of RAB7 + (F) and LAMP1 + (G) areas per cell in control and MYCT1 KD cells. n = 3 independent experiments; 20–50 cells were analyzed per condition for each experiment; mean ± SD; Welch’s t test, P > 0.05. (H) MYCT1 knockdown increased FITC-dextran uptake. 2 days after siRNA transfection, cells were starved for 1 h in PBS, followed by a 30-min induction with amino acid solution together with 10-kDa FITC dextran. Detection of 10-kDa FITC-dextran (gray) and staining of ECs for VE-cadherin (magenta) and DAPI (blue). Arrow, dextran + puncta. Scale bar, 10 μm. (I) Quantification of the number of dextran + puncta per cell in control and MYCT1 KD cells. n = 3 independent experiments; 30–50 cells were analyzed per condition for each experiment; mean ± SD; Welch’s t test, P = 0.0016 (*). (J) Example of gating strategy (7-AAD neg CD45 neg CD31 + ) of ECs from gonadal fat pad by flow cytometry. (K) WAT ECs take up higher amounts of labeled plasma proteins compared with colon ECs. Quantification of labeled plasma protein uptake in ECs from s.c. and visceral WAT and colon normalized to plasma Atto-647 signal. n = 10 mice per organ; Friedman test with Dunn’s multiple comparisons test, P > 0.05 for scFAT versus visFAT, P = 0.0052 for scFAT versus colon, and P = 0.001 (*) for visFAT versus colon. (L) Endocytosis inhibition with dynasore rescues mTORC1 hyperactivation caused by knockdown of MYCT1 . Staining for p-S6 (gray), β-catenin (magenta), and DAPI (blue). Scale bar, 50 μm. (M) Quantification of mTORC1 activation by amino acid supplementation in control and MYCT1 KD cells in the absence or presence of dynasore. The percentage of p-S6 + cells was quantified in the indicated conditions. n = 3 independent experiments; 1,500–6,000 cells were analyzed per condition for each experiment; mean ± SD; two-way ANOVA with Tukey’s multiple comparisons test, P = 0.004 (*) for MYCT1 knockdown effect in control conditions and P < 0.001 (*) for its rescue by dynasore treatment. (N) RAB5 knockdown rescues mTORC1 hyperactivation in MYCT1 KD cells. Staining of ECs for p-S6 (gray), β-catenin (magenta), and DAPI (blue). Scale bar, 50 μm. (O) Quantification of mTORC1 activation in control, MYCT1 KD , and MYCT1-RAB5 KD cells. The percentage of p-S6 + cells was quantified in the indicated conditions. n = 3 independent experiments; 7,000-15,000 cells were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Tukey’s multiple comparisons test, P = 0.0021 (*) for MYCT1 knockdown effect and P = 0.0292 (*) for its rescue by RAB5 double knockdown. (P) IFITM2/3 knockdown rescues mTORC1 hyperactivation in MYCT1 -deficient human adipose ECs. Staining for p-S6 (gray), β-catenin (magenta), and DAPI (blue). Scale bar, 100 μm. (Q) Quantification of mTORC1 activation in control, MYCT1 KD , IFITM2/3 KD , and MYCT1 – IFITM2/3 KD cells. The percentage of p-S6 + cells was quantified in the indicated conditions. n = 2 independent experiments; 1,500–3,000 cells were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Tukey’s multiple comparisons test, P = 0.0168 (*) for MYCT1 knockdown effect and P = 0.0123 (*) for rescue effect by IFITM2/3 double knockdown.

Article Snippet: IFITM3 (human) reverse , Eurofins , 5′-CCA GCA CAG CCA CCT CG-3′.

Techniques: Activation Assay, Biomarker Discovery, Knockdown, Staining, Control, Transfection, Flow Cytometry, Labeling, Clinical Proteomics, Inhibition