Journal: The Journal of Biological Chemistry

Article Title: MARCH8-mediated ubiquitination regulates expression of the antiviral protein IFITM3

doi: 10.1016/j.jbc.2025.110879

Figure Lengend Snippet: Identification of IFITM3 as an interactor of MARCH8. A, schematic representation of the identification process. HeLa cells were transfected with MARCH8 plasmid or empty vector. Cells were then treated with lysosomal inhibitor chloroquine (CQ). Potential interactors of MARCH8 were identified by LC–MS/MS analysis of MARCH8 co-IP protein samples. The empty vector group was used as a negative control. B, KEGG pathway bubble diagram of top 20 differentially expressed proteins. The rich factor represents the enrichment factor, which is the ratio of the proportion of differentially expressed proteins annotated to the pathway to the proportion of background proteins annotated to the pathway. C, COG function classification of unigenes shows 25 different functional groups. D, differential proteins were nalysed according to mass spectrum score (score) and iBAQ protein intensity. Proteins known to interact with MARCH8 are highlighted in light blue . E and F, reciprocal co-IP analysis showed the interaction between MARCH8 and IFITM3 in HEK293T cells. G, co-IP analysis showed the interaction between MARCH8 and endogenous IFITM3 in HeLa cells. H, HeLa cells were transfected with FLAG-MARCH8 or empty vector plasmid. Cells were treated with CQ (50 μM) for 4 h and processed for immunofluorescence staining with anti-IFITM3 and anti-FLAG antibodies. Scale bars indicate 5 μm in all panels. Statistical result of the colocalization analysis is shown on the right . Values are means ± SD from >50 cells (n = 3 independent experiments). Statistical differences were determined by two-sided Student’s t test. I and J, co-IP and Western blotting analysis of HEK293T cells were performed after transfection with plasmids encoding FLAG-IFITM3 and full-length MARCH8-Myc or its truncation mutants ( I ) or with plasmids encoding MARCH8-Myc and full-length FLAG-IFITM3 or its truncation mutants ( J ). Schematic diagrams of MARCH8 ( I ) and IFITM3 ( J ) and their truncation mutants are shown above the Western blots. COG, clusters of orthologous groups of proteins; co-IP, coimmunoprecipitation; HEK293T, human embryonic kidney 293T cell line; iBAQ, intensity-based absolute quantification; IFITM3, interferon-induced transmembrane protein 3; KEGG, Kyoto Encyclopedia of Genes and Genomes; MARCH, Membrane-Associated RING-CH.

Article Snippet: Antibodies used for Western blot are as follows: MARCH8 (Proteintech; catalog no.: 14119-1-AP; 1:1000 dilution), IFITM3 (Proteintech; catalog no.: 11714-1-AP; 1:1000dilution), FLAG (Sigma–Aldrich; catalog no.: F7425; 1:2000 dilution), Myc (Sigma–Aldrich; catalog no.: C3956; 1:2000 dilution), HA.11 Epitope Tag (BioLegend; catalog no.: 901501 or catalog no.: 902301; 1:1000 dilution), and actin (Sigma–Aldrich; catalog no.: A1978; 1:2000 dilution).

Techniques: Transfection, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, Negative Control, Functional Assay, Immunofluorescence, Staining, Western Blot, Quantitative Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: MARCH8-mediated ubiquitination regulates expression of the antiviral protein IFITM3

doi: 10.1016/j.jbc.2025.110879

Figure Lengend Snippet: Effects of MARCH8 on IFITM3 degradation. A, . HEK293T cells were cotransfected with FLAG-IFITM3 and WT MARCH8 (WT) or a control vector, followed by Western blot. Values are means ± SD from three independent experiments (n = 3). Statistical differences were determined by two-sided Student’s t test; ∗∗∗ p < 0.0001. B, HEK293T cells were cotransfected with FLAG-IFITM3 and increasing amounts of MARCH8, followed by Western blot. Values are means ± SD from three independent experiments (n = 3). Statistical differences were determined by two-sided Student’s t test; ∗ p < 0.01, ∗∗ p < 0.001. C, HEK293T cells were transfected with WT MARCH8 (WT) or a control vector, followed by Western blot. Values are means ± SD from three independent experiments (n = 3). Statistical differences were determined by two-sided Student’s t test; ∗∗ p < 0.001. D – H, HeLa cells were transfected with siRNA targeting MARCH8 or control siRNA. MARCH8 and IFITM3 expression was examined by Western blotting ( D ). IFITM3 expression was also examined by flow cytometry assay ( E ) and immunofluorescence ( G ). Scale bars indicate 20 μm in all panels. Values are means ± SD from three independent experiments (n = 3). Statistical differences were determined by two-sided Student’s t test; ∗∗∗ p < 0.0001, ∗∗ p < 0.001. I – K, the endogenous MARCH8 in A549 ( I ) and HeLa ( J ) cells was knocked out by lentiviral CRISPR–Cas9. MARCH8 and IFITM3 expression was examined by Western blotting ( I and J ). IFITM3 expression in HeLa cells was also examined by immunofluorescence ( K ). The quantification is shown on the right . Scale bars indicate 50 μm in all panels. Values are means ± SD from three independent experiments (n = 3). Statistical differences were determined by two-sided Student’s t test; ∗∗∗ p < 0.0001, ∗∗ p < 0.001. HEK293T, human embryonic kidney 293T cell line; IFITM3, interferon-induced transmembrane protein 3; MARCH, Membrane-Associated RING-CH.

Article Snippet: Antibodies used for Western blot are as follows: MARCH8 (Proteintech; catalog no.: 14119-1-AP; 1:1000 dilution), IFITM3 (Proteintech; catalog no.: 11714-1-AP; 1:1000dilution), FLAG (Sigma–Aldrich; catalog no.: F7425; 1:2000 dilution), Myc (Sigma–Aldrich; catalog no.: C3956; 1:2000 dilution), HA.11 Epitope Tag (BioLegend; catalog no.: 901501 or catalog no.: 902301; 1:1000 dilution), and actin (Sigma–Aldrich; catalog no.: A1978; 1:2000 dilution).

Techniques: Control, Plasmid Preparation, Western Blot, Transfection, Expressing, Flow Cytometry, Immunofluorescence, CRISPR, Membrane

Journal: The Journal of Biological Chemistry

Article Title: MARCH8-mediated ubiquitination regulates expression of the antiviral protein IFITM3

doi: 10.1016/j.jbc.2025.110879

Figure Lengend Snippet: MARCH8 leads to IFITM3 degradation in lysosomes through the K63-linked polyubiquitylation . A and B, MARCH8 WT and knockdown (KD) HeLa cells were treated by CHX (100 μM) for 0, 2, 4, and 6 h followed by Western blot. Lanes shown are nonadjacent but from the same immunoblot. The quantification is shown on the right ( B ). Quantifications derive from three independent experiments (mean ± SD). C, HEK293T cells were cotransfected with FLAG-IFITM3 and MARCH8 WT or empty vector. Cells were then treated with DMSO, MG132 (50 μM), bafilomycin (1 μM), CQ (50 μM), and 3-MA (100 μM) for 4 h and processed for Western blot. D, HEK293T cells were cotransfected with FLAG-IFITM3 and WT MARCH8 (WT), the W114A mutant MARCH8 (W114A), or the Y222A mutant MARCH8 (Y222A) followed by Western blot. E, statistical results of the Western blot ( D ). Values are means ± SD from three independent experiments (n = 3). Statistical differences were determined by two-sided Student’s t test; ∗∗∗ p < 0.0001. ns, nonsignificant. F , HEK293T cells were cotransfected for 24 h with HA-Ub, FLAG-IFITM3, and vector, MARCH8, or NEDD4. Cells were treated with CQ (50 μM) for 4 h. Cells were harvested for co-IP using anti-FLAG Ab-coated agarose beads and analyzed by Western blot. G, HEK293T cells were cotransfected for 24 h with HA-Ub, FLAG-IFITM3, and vector, MARCH8 WT, or MARCH8 W114A. Cells were treated with CQ (50 μM) for 4 h. Cells were harvested for co-IP using anti-FLAG beads and analyzed by Western blot. H, HEK293T cells were transfected with HA-tagged ubiquitin mutants, FLAG-IFITM3 and MARCH8. Cells were treated with CQ (50 μM) for 4 h. Cell lysates were subject to IP with anti-FLAG antibody, and the IP and input samples were analyzed by Western blot with antibodies against the indicated protein targets. Ab, antibody; CHX, cycloheximide; co-IP, coimmunoprecipitation; CQ, chloroquine; DMSO, dimethyl sulfoxide; HEK293T, human embryonic kidney 293T cell line; IFITM3, interferon-induced transmembrane protein 3; K63, lysine 63; MARCH, Membrane-Associated RING-CH; Ub, ubiquitin.

Article Snippet: Antibodies used for Western blot are as follows: MARCH8 (Proteintech; catalog no.: 14119-1-AP; 1:1000 dilution), IFITM3 (Proteintech; catalog no.: 11714-1-AP; 1:1000dilution), FLAG (Sigma–Aldrich; catalog no.: F7425; 1:2000 dilution), Myc (Sigma–Aldrich; catalog no.: C3956; 1:2000 dilution), HA.11 Epitope Tag (BioLegend; catalog no.: 901501 or catalog no.: 902301; 1:1000 dilution), and actin (Sigma–Aldrich; catalog no.: A1978; 1:2000 dilution).

Techniques: Knockdown, Western Blot, Plasmid Preparation, Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Ubiquitin Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: MARCH8-mediated ubiquitination regulates expression of the antiviral protein IFITM3

doi: 10.1016/j.jbc.2025.110879

Figure Lengend Snippet: K24 is the primary ubiquitination site of IFITM3 by MARCH8 . A, amino acid sequences of IFITM3 and the indicated mutants. Lysine residues (K)/arginine residue (R) mutants are marked as red . B, HEK293T cells were transfected with FLAG-IFITM3 mutants and vector or MARCH8 WT followed by Western blot. C, HEK293T cells were transfected with FLAG-IFITM3 WT or 4KR mutant and vector, MARCH8, or NEDD4 followed by Western blot. D, HeLa cells were cotransfected with FLAG-IFITM3 WT or 4KR mutant and vector or MARCH8. Cells were treated with CQ (50 μM) for 4 h and processed for immunofluorescence staining with anti-FLAG and anti-MARCH8 antibodies. Scale bars indicate 5 μm in all panels. E, ubiquitination of IFITM3 mutants by MARCH8. HEK293T cells were transfected with FLAG-IFITM3 mutants, HA Ub WT, and MARCH8. Cells were treated with CQ (50 μM) for 4 h. Cell lysates were subject to IP with anti-FLAG antibody, and the IP and input samples were analyzed by Western blotting with antibodies against the indicated protein targets. F, HEK293T cells were cotransfected with MARCH8 WT and FLAG-IFITM3 WT, FLAG-IFITM3 D1-21 mutants, or FLAG-IFITM3 YLAA mutants for Western blots. G, HeLa cells were cotransfected with MARCH8 WT and FLAG-IFITM3 WT, D1-21 mutants, or YLAA mutants. Cells were treated with CQ (50 μM) for 4 h and processed for immunofluorescence staining with anti-FLAG and anti-MARCH8 antibodies. Scale bars indicate 5 μm in all panels. H, HEK293T cells were transfected with FLAG-IFITM3 mutants D1-21 and YLAA, HA-Ub WT, and MARCH8. Cell lysates were subject to IP with anti-FLAG antibody, and the IP and input samples were analyzed by Western blotting with antibodies against the indicated protein targets. CQ, chloroquine; HEK293T, human embryonic kidney 293T cell line; IFITM3, interferon-induced transmembrane protein 3; IP, immunoprecipitation; K24, lysine 24; MARCH, Membrane-Associated RING-CH.

Article Snippet: Antibodies used for Western blot are as follows: MARCH8 (Proteintech; catalog no.: 14119-1-AP; 1:1000 dilution), IFITM3 (Proteintech; catalog no.: 11714-1-AP; 1:1000dilution), FLAG (Sigma–Aldrich; catalog no.: F7425; 1:2000 dilution), Myc (Sigma–Aldrich; catalog no.: C3956; 1:2000 dilution), HA.11 Epitope Tag (BioLegend; catalog no.: 901501 or catalog no.: 902301; 1:1000 dilution), and actin (Sigma–Aldrich; catalog no.: A1978; 1:2000 dilution).

Techniques: Ubiquitin Proteomics, Residue, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Immunofluorescence, Staining, Immunoprecipitation, Membrane

Journal: The Journal of Biological Chemistry

Article Title: MARCH8-mediated ubiquitination regulates expression of the antiviral protein IFITM3

doi: 10.1016/j.jbc.2025.110879

Figure Lengend Snippet: MARCH8 regulates IFITM3 turnover, localization, and trafficking. A – D, HeLa cells were transfected with FLAG-IFITM3 and vector or EGFP-MARCH8, then permeabilized, costained with FLAG and EEA1 antibodies ( A ) or LAMP1 antibodies ( B ). Statistical result of the colocalization analysis is shown on the figures ( C and D ). Scale bars indicate 5 μm in all panels. Values are means ± SD from >50 cells (n = 3 independent experiments). Statistical differences were determined by two-sided Student’s t test; ∗∗ p < 0.001, ns, nonsignificant. E – H, HeLa cells were transfected with siRNA targeting MARCH8 or control siRNA, then costained with IFITM3 and EEA1 antibodies ( E ) or LAMP1 antibodies ( F ). Statistical result of the colocalization analysis is shown on the figures ( G and H ). Scale bars indicate 5 μm in all panels. Values are means ± SD from >50 cells (n = 3 independent experiments). Statistical differences were determined by two-sided Student’s t test; ∗∗∗ p < 0.0001, ∗∗ p < 0.001. I, MARCH8 WT and KO HeLa cells were treated by IFN-α (250 μM) or DMSO. After 24 h, the cells were treated by CHX (100 μM) for 0, 3, and 6 h followed by Western blot. The quantification is shown on the right and derives from three independent experiments (mean ± SD; unpaired t test). Statistical differences were determined by two-sided Student’s t test; ∗∗ p < 0.001, ∗ p < 0.01. J – Q, MARCH8 WT and KO HeLa cells were treated by DMSO ( J , K ) or IFN-α ( N , O ), then costained with IFITM3 and EEA1 antibodies ( J , N ) or LAMP1 antibodies ( K , O ). Statistical result of the colocalization analysis is shown on the figures ( L , M and P , Q ). Scale bars indicate 5 μm in all panels. Values are means ± SD from >50 cells (n = 3 independent experiments). Statistical differences were determined by two-sided Student’s t test; ∗∗∗ p < 0.0001, ns, nonsignificant. CHX, cycloheximide; DMSO, dimethyl sulfoxide; EGFP, enhanced GFP; HEK293T, human embryonic kidney 293T cell line; IFITM3, interferon-induced transmembrane protein 3; IFN-α, interferon alpha; MARCH, Membrane-Associated RING-CH.

Article Snippet: Antibodies used for Western blot are as follows: MARCH8 (Proteintech; catalog no.: 14119-1-AP; 1:1000 dilution), IFITM3 (Proteintech; catalog no.: 11714-1-AP; 1:1000dilution), FLAG (Sigma–Aldrich; catalog no.: F7425; 1:2000 dilution), Myc (Sigma–Aldrich; catalog no.: C3956; 1:2000 dilution), HA.11 Epitope Tag (BioLegend; catalog no.: 901501 or catalog no.: 902301; 1:1000 dilution), and actin (Sigma–Aldrich; catalog no.: A1978; 1:2000 dilution).

Techniques: Transfection, Plasmid Preparation, Control, Western Blot, Membrane

Journal: The Journal of Biological Chemistry

Article Title: MARCH8-mediated ubiquitination regulates expression of the antiviral protein IFITM3

doi: 10.1016/j.jbc.2025.110879

Figure Lengend Snippet: MARCH8 KO increases IFITM3 level and protects cells from virus entry . A – C, MARCH8 WT and KO A549 cells were transduced with 100 or 200 ng p24/ml VSV G pseudoviral particles (VSV G pp) for 48 h. Half of the cells were then fixed and examined for GFP positivity to measure the percentage of cells infected using flow cytometry ( A and B ), the other half of the cells were lysed to measure luciferase activity ( C ). Values are means ± SD from three independent experiments (n = 3). Statistical differences were determined by two-sided Student’s t test; ∗∗∗ p < 0.0001. D and E, MARCH8 WT and KO HeLa cells were transfected with vector, MARCH8 WT, or W114A for 24 h and then transduced with VSV G pp for 48 h. The cells were lysed; luciferase activity was measured to report viral infection ( D ). MARCH8 levels and the effect of MARCH8 WT and W114A on endogenous IFITM3 levels were analyzed by Western blot ( E ). Values are means ± SD from three independent experiments (n = 3). Statistical differences were determined by two-sided Student’s t test; ∗∗∗ p < 0.0001, ∗∗ p < 0.001. ns, nonsignificant. F – J, MARCH8 WT and KO A549 cells were incubated with HIV-luc pseudoviral virus particles with the indicated viral envelope proteins. HA proteins from various influenza A virus strain include H1 (PR): A/PR/8/34 (H1N1), H3 (Udorn): A/Udorn/72 (H3N2), H5 (Thai): A/Thailand2 (SP-33)/2004 (H5N1), H7 (FPV): A/FPV/Rostock/34 (H7N1). Luciferase activity was measured to report viral infection. Values are means ± SD from three independent experiments (n = 3). Statistical differences were determined by two-sided Student’s t test; ∗∗∗ p < 0.0001, ∗∗ p < 0.001, and ∗ p < 0.01. IFITM3, interferon-induced transmembrane protein 3; MARCH, Membrane-Associated RING-CH; VSV G, vesicular stomatitis virus glycoprotein.

Article Snippet: Antibodies used for Western blot are as follows: MARCH8 (Proteintech; catalog no.: 14119-1-AP; 1:1000 dilution), IFITM3 (Proteintech; catalog no.: 11714-1-AP; 1:1000dilution), FLAG (Sigma–Aldrich; catalog no.: F7425; 1:2000 dilution), Myc (Sigma–Aldrich; catalog no.: C3956; 1:2000 dilution), HA.11 Epitope Tag (BioLegend; catalog no.: 901501 or catalog no.: 902301; 1:1000 dilution), and actin (Sigma–Aldrich; catalog no.: A1978; 1:2000 dilution).

Techniques: Virus, Transduction, Infection, Flow Cytometry, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Incubation, Membrane