Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) E2012 and E2012-BPyne structures. (b) Cell membranes were photolabeled with E2012-BPyne and then clicked with TAMRA-azide and analyzed (left: Coomassie blue; right: in-gel fluorescence). (c) Biotinylated proteins were captured by E2012-BPyne and analyzed by WB. (d) IFITM3 co-immunoprecipitated with γ-secretase subunits using anti-PS1. (e) IFITM3 was co-purified with γ-secretase subunits by GY6 or 163-BP-L-biotin. (f) An interaction (red) between PS1 and IFITM3 was determined by in situ PLA in mouse primary neurons (bottom panel). Negative controls using PS1 or IFITM3 alone (top two panels), dapi (blue), F-actin (green), scale bar = 50μm. (g) Labeling of IFITM3 by E2012-Bpyne in WT and dKO MEF cells. All WB and IF images are representative of three independent experiments (except b: 2 replicates).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Fluorescence, Immunoprecipitation, Purification, In Situ, Labeling

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) LC-MS/MS analysis of the 15 kDa band identified four peptides that match with human IFITM3. (b) WB analysis of E2012-BPyne (500 nM) labeled PS1-NTF protein. (c) Structures of imidazole GSMs, acid GSM and GSIs. (d) WB analysis of E2012-BPyne labeled proteins in the absence or presence of imidazole GSMs, acid GSM and GSIs. Labeled proteins were captured and analyzed by WB for IFITM3. (e) Structures of GY6 and 163-BP-L-biotin. (f) IFITM3 co-immunoprecipitates with γ-secretase subunits. CHAPSO solubilized cell membranes were immunoprecipitated with anti-IFITM3 antibody and probed with antibodies against PS2-CTF and Pen-2. Rabbit IgG was used as a negative control. (g) IFITM3 does not co-immunoprecipitate with SPP. CHAPSO solubilized cell membranes were immunoprecipitated with a monoclonal anti-IFITM3 antibody (9D11) and probed with antibodies against SPP and IFITM3. Mouse IgG was used as a negative control. (h) Analysis of the total protein level in WT MEF or PS1/2 double KO MEF cells. The same amount of membrane proteins was loaded and analyzed by Western blotting. ( i ) IFITM3 mRNA expression levels were measured by RT-PCR in WT MEF or PS1/2 double KO MEF cells (n=6). All WB images and graphs are representative of three independent experiments (except; a/d: 2 replicates). Graphs are mean ± SD. ns, not significant, two-sided Student’s t-test.

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Liquid Chromatography with Mass Spectroscopy, Labeling, Immunoprecipitation, Negative Control, Membrane, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) IFITM3 KD by siRNA (n=3) in HEK-APP WT cells was confirmed by WB. Scramble siRNA (SC, n=3), a negative control. (b) IFITM3 KD reduces secreted Aβ40 (n=6, ****p<0.0001) and 42 (n=6, ****p<0.0001). Aβ levels were calculated as % of SC. (c) IFITM3 was KO of U138 cells, and empty vector (EV) was used as control. IFITM3 was reintroduced by transient transfection in both EV and KO cell lines. IFITM3 expression was confirmed by WB. (d) Effect of KO and rescue of IFITM3 on γ-secretase activity for Aβ40 (n=3, *p= 0.0249, ****p<0.0001) and 42 (n=3, *p=0.0359, **p=0.0025) cleavage. (e) Comparison IC 50 of GSM E2012-BPyne against γ-secretase for Aβ40 (n=6, **p= 0.0017) and Aβ42 (n=6, ns) cleavages in U138 EV and KO cells. All WB images and graphs are representative of three independent experiments (except; e: contains data from 2 experimental replicates of n=3). Graphs are mean ± SD. Ns, not significant, two-sided Student’s t-test (except; d: one-way ANOVA and Fishers LSD test).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Negative Control, Plasmid Preparation, Control, Transfection, Expressing, Activity Assay, Comparison

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Quantification of WB showed that IFITM3 KD did not change protein expression levels of APP, Nct and PS1-NTF in HEK-APP WT cells (n=3). (b) Schematic representation of cell-free γ-secretase assay. γ-Secretase is incubated with a recombinant APP substrate in the presence of 0.25% CHAPSO. Cleaved Aβ40 and 42 species are measured with cleavage specific antibodies and AlphaLISA technology. ( c ) Schematic model showing different GSM and GSI binding sites in γ-secretase: E2012 (imidazole GSM), GSM-1 (acid GSM), and L458 (transition state analogue inhibitor, GSI). (d-f) Comparison of IC 50 of (d) GSM-25 (EV: n=9, KO: n= 8) for Aβ40 (****p<0.0001) and Aβ42 (ns), (e) GSM-1 (n=6) for Aβ40 (**p=0.0039) and Aβ42 (ns), and (f) L458 (n=3) for Aβ40 (ns) and Aβ42 (ns) cleavages in the U138 EV or KO cell lines (n≥3). (g) IFITM3 knockdown (KD) does not affect expression of γ-secretase subunits. IFITM3 was knocked-down by siRNA (6 pmol, n=3) in HEK-NotchΔE cells and scramble siRNA (SC, n=3) was used as a negative control. Cell lysates were probed by antibodies against Nct, PS1-NTF and IFITM3. β-Actin was used as a loading control. (h) Effect of IFITM3 KD on γ-secretase activity. IFITM3 KD increased γ-secretase cleaved product NICD, analyzed by WB. Cell lysates were probed by antibodies against c-myc (NotchΔE) and NICD and a representative quantification of NICD (n=8, ***p=0.001) is shown (lower panel). (i) Cell based NICD AlphaLISA assay (left panel) revealed an increase in NICD production with IFITM3 KD. Quantification of NICD (n=8, ***p=0.001) is shown in the right panel. (j) Effect of IFITM3 KO on γ-secretase activity. KO cells lines have increased γ-secretase activity as compared to the EV cell line. The NICD cleavage in vitro was measured by AlphaLISA assay (n=3,**p=0.0096). All WB images and graphs are representative of three independent experiments Graphs are mean ± SD. ns, not significant, two-sided Student’s t-test.

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Expressing, Incubation, Recombinant, Binding Assay, Comparison, Knockdown, Negative Control, Control, Activity Assay, In Vitro

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Protein levels of γ-secretase subunits and IFITM3 in pooled membranes from 4- and 28-month-old WT mouse brains (n=5 mice per age and sex group except n=4 for 28 female, female: **p=0.0014, male: **p=0.0065). Quantified as percent of 4-month female level. (b) γ-Secretase activity for Aβ40 (female: p=***0.0002, male: **p=0.0095) and Aβ42 (female: p=**0.0024, male: **p=0.0088) production in vitro from pooled membrane. (c) Solubilized membranes were captured by 163-BP-L-biotin and then analyzed for γ-secretase and IFITM3 levels. (d) WB for PS1-NTF and IFITM3 (left panel, representative mice shown) in membranes of 18-month-old WT (n=4) and IFITM3−/− (n=5) mouse brains. γ-Secretase activity for Aβ40 (***p=0.0004) and 42 (****p=<0.0001) cleavage (right panels). (e) APP, Nct, PS1-NTF and IFITM3 in membranes from 3- and 12-month-old WT and 5xFAD mice. Quantified as percent of 3-months-old WT (n=5 per group except n=4 for WT at 12 months)(3moWT-12mo5X: ****p<0.0001, 12moWT-12mo5X: ***p=0.004, 3mo5X-12mo5X: ***p=0.0003). (f) WB for PS1-NTF and IFITM3 (left panel) in membranes from 4-month-old 5xFAD (n=3) and IFITM3−/−; 5xFAD (n=3) mouse brains. γ-Secretase activity in vitro for Aβ40 (***p=0.0003) and 42 (**p=<0.0011) cleavage (right panels). (g) Fluorescence microscopy of amyloid plaques in cortex and hippocampus (Thioflavin-S, green) in 4-month-old PFA perfused mice (5xFAD: n=4, IFITM3−/−; 5xFAD: n=5). Scale bar = 300μm (left), 200μm (right). Number of plaques per mm 2 of tissue was calculated throughout the brain and averaged (cor (cortex): *p=0.0109, hip (hippocampus): **p=0.0026). All WB and IF images and graphs are representative of three independent experiments (except; c and quantifications in e: 2 replicates and g: every 1/6 th section throughout the brain). Bar graphs are mean ± SD. Violin plots represent median (middle line) and interquartile range (outer lines). Ns, not significant, two-sided Student’s t-test (except; e: two-way ANOVA followed by Tukey).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Activity Assay, In Vitro, Membrane, Fluorescence, Microscopy

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) WBs for Nct and PS1-NTF were quantified by Odyssey imaging (n=5 mice pooled per group, except n=4 for 28F, all=ns). (b) Effect of aging on subcellular localizations of IFITM3. A hemibrain from male wild-type C57BL/6 mouse at 4 and 28 months (n=1 per group) were homogenized and layered on iodixanol gradient (2.5 – 30 %). Fractions were collected from the top and resolved by WBs for γ-secretase, IFITM3 and different subcellular markers. (c) WBs for APP, Nct, and PS1-NTF were quantified by Odyssey imaging (n=5 mice per group except n=4 for WT at 12 months). APP: 3moWT-3mo5X: ****p<0.001, 3mo5X-12mo5X: ****p<0.0001, 12moWT-12mo5X: ****p<0.0001). Nct: 3moWT-12moWT: ****p<0.001, 3mo5X-12mo5X: ****p<0.001, 12moWT-12mo5X: ****p<0.001. PS1-NTF: 3moWT-12mo5X: ***p=0.0004, 12moWT-12mo5X: ****p<0.001. ( d ) Immunostaining of IFITM3 in mouse brains. Fluorescence microscopy of IFITM3 expression in 12-month-old PFA perfused mice (WT, upper panel, 5XFAD, lower panel). Representative images of cortex, hippocampus and subiculum (left to right) show IFITM3 (green) and DAPI (blue). Scale bar = 1000μm, 200μm, 100μm (left to right). Total IFITM3 fluorescence area within the hippocampus and cortex of WT and 5XFAD was quantified using FIJI. Total IFITM3 was divided by tissue area and 5XFAD expression was normalized to average of WT (WT: n=7, 5XFAD: n=9)(cor (cortex): **p=0.0035, hip (hippocampus): ****p<0.0001 ). (e) IFITM3 expression in astrocytes and microglia is upregulated in 5XFAD mice compared to WT mice. Fluorescence microscopy of IFITM3, GFAP (top) and Iba1 (bottom) expression in 12-month-old PFA perfused mice (WT, upper panel, 5XFAD, lower panel). Representative images of the hippocampus and cortex show IFITM3 (red), GFAP (green – top), Iba1 (green - bottom), and DAPI (blue), scale bar = 500μm. Inset panels (left to right) show GFAP or Iba1 (green), IFITM3 (red) and merge. Scale bar = 50μm. All WB images and graphs are representative of three independent experiments (except; b: 2 replicates). Graphs are mean ± SD. ns, not significant, , two-sided Student’s t-test, (except; c: one-way ANOVA followed by Tukey).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Imaging, Immunostaining, Fluorescence, Microscopy, Expressing

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) The expression profiles of IFITM3 in human control (n=76) and LOAD (n=80) from temporal cortex (**p=0.002)(Mayo Clinic cohort). (b) IFITM3 mRNA expression levels in control and LOAD samples (LOAD: n=18 and control: n=10, **p=0.0056). (c) The protein levels of γ-secretase subunits and IFITM3 in human brain membranes (LOAD: n=18 and control: n=10, *p=0.0127). IFITM3 quantified as relative intensity. (d) IFITM3 expression (left) between control (n=10), LOAD-L (n=10) and LOAD-H (n=8)(****p<0.0001). γ-Secretase activity for Aβ40 (**p=0.0037, ***p=0.0007) and 42 (**p=0.0036, ***p=0.0002) cleavage between groups. (e) Primary mouse neurons were treated with control (n=2), 10ng/mL (n=3, ***p=0.0005) or 100 ng/mL (n=3, ***p=0.0003) of IFN-γ, membranes were probed for γ-secretase and IFITM3 (left panel, quantified: right panel). (f) Quantification of secreted Aβ40 from 10ng/mL (n=8, **p=0.0010) and 100 ng/mL (n=8, **p=0.0026) IFN-γ treated neurons Aβ42 from 10ng/mL (n=8, ***p=0.0006) and 100 ng/mL (n=8, ***p=0.0003) IFN-γ (n=8). (g) γ-Secretase activity for Aβ40 (n=6, ***p=0.0007) and 42 (n=6, ***p=0.006) cleavage from IFN-γ treated neuron membranes. (h) Photolabeled PS-NTF in neuronal membranes and quantified as % of control (control: n=10, 10ng/ml: n=5, **p=0.0023, 100ng/ml: n=7,****p<0.0001). (i) WB for IFITM3 and PS1-NTF primary human astrocytes treated with PBS, IL-6, or IL-1β (control: n=6, IL-6: n=4, **p=0.0014, IL-1β: n=3, *p=0.0103), data normalized to PBS. (j) γ-Secretase activity for Aβ40 (control: n=8 IL-6: n=7, **p=0.0098, IL-1β: n=9,****p<0.0001) and 42 (control: n= IL-6: n=7, *p=0.0467, IL-1β: n=9, ****p<0.0001) in astrocyte membrane. All WB images and graphs are representative of three independent experiments (except; b: 1 replicate, c/g: 2 replicates, d: γ-secretase activity graph contains data from two independent replicates, h: 5 replicates). Graphs are mean ± SD. Ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-sided Student’s t-test.

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Expressing, Control, Activity Assay, Membrane

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Spearman’s correlation of mRNA expression of human IFITM3 gene with age was analyzed in the cortex (n=158) and hippocampus (n=123) of normal human brains using the Genotype-Tissue Expression (GTEx) cohort.. (b) mRNA expression in non-demented subject control (n=10) and LOAD samples (n=18) of MAP2 (ns), GFAP (**p=0.0046), and AIF1 (ns) were measured, which were used in – . (c) Expression profiles of MAP2 (ns), GFAP (****p<0.0001), and AIF1 (ns) in the temporal cortex of human control (n=76) and LOAD samples (n=80) using the Mayo Clinic cohort data. Correlation analyses were carried out and p values were calculated. The protein levels of Nct (****p<0.0001) and PS1-NTF (**p=0.0042) in human brain membranes (control and LOAD). The samples were analyzed by WB and quantified (n=10 and 18, respectively). Signal was normalized to HeLa cell membrane. (e-f) IFITM3 SNP Genotypes. (e) Allelic discrimination plot depicting rs34481144 genotype calls for control (n=9), LOAD-L (n=10), and LOAD-H (n=8) brain samples. The axes show delta Rn values obtained from TaqMan SNP genotyping analysis. Samples without genomic DNA were used as non-template controls (shown as black squares in the left lower quadrant, n=2). (f) Allele frequency of rs34481144 genotype in control (n=9) and LOAD (n=18). ( g ) mRNA level of IFITM3 gene in four types of EGFP/L10a-expressing mouse hippocampal neurons (GAD2 (glutamate decarboxylase 2), CCK (cholecystokinin), PV (parvalbumin), and CORT (cortistatin) expressing GABAergic neurons)(n=4 per group, GAD2-PV: ***p=0.0003, CCK-PV: ****p<0.0001, CCK-Cort *p=0.0363, PV-Cort: **p=0.0059). ( h ) mRNA levels of IFITM3 in human iPSC-derived neurons (n=4) and human primary astrocytes (n=3) were measured by qPCR (****p<0.0001). ( i-j ) Human iPSC-derived neurons (i) and human primary astrocytes (j) were stained for IFITM3 with MAP2 (neuronal marker) or S100β (astrocyte marker). DAPI was used for nucleus staining. Scale bar = 200 and 500 μm. ( k ) Induction of IFITM3 by IFN-α in primary neurons. E16 mouse primary neurons were treated with 100 ng/ml of IFN-α at DIV12 for 24 hours. The protein levels of γ-secretase and IFITM3 were analyzed by WB (n=4 per group). β-Tubulin III was used as a loading control. ( l ) Effect of IFITM3 induction on γ-secretase activity for Aβ40 (*p=0.0116) and Aβ42 (*p=0.0319) activity. Membranes from primary neurons were incubated with the recombinant APP substrate C100-ΔID-FLAG and γ-secretase activity (Aβ cleavage rate) was assayed by human Aβ three-plex MSD kits (n=12, 10). ( m ) JC8 whole cell photolabeling. Neuronal membranes were photolabeled with JC8 in the absence or presence of L458 and analyzed by anti-PS1-NTF antibody. Photolabeled PS1-NTF protein level was quantified by Odyssey imaging (n=3, *p=0.0210). (n) Spearman’s correlation between the expression level of IFITM3 and viruses. In the Brodmann Area 22 (BA-22 region) in the Mount Sinai Brain Bank (MSBB) cohort, the expression level of IFITM3 is positively correlated with the expression level of the human herpesvirus-6B (HHV-6B) (rho=0.248, p=0.044, n=66). In the Brodmann Area 36 (BA-36 region), the expression level of IFITM3 is positively correlated with the expression of hepatitis C virus genotype 4 (rho=0.255, p=0.033, n=70). All WB images and graphs are representative of two independent experiments (except; b/e: 1 replicate, h: data pooled from 2 experiments, k: 3 replicates, l: data pooled from 4 experiments). Bar graphs are mean ± SD. Violin plots represent median (middle line) and interquartile range (outer lines). ns, not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001, two-sided Student’s t-test (except; g: One-Way ANOVA followed by Tukey).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Expressing, Control, Membrane, Derivative Assay, Staining, Marker, Activity Assay, Incubation, Recombinant, Imaging, Virus

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Schematic model showing L458 binding to the subsites (S2-S3’) in the active site of γ-secretase. (b) Photolabeling of IFITM3 and PS1 by four inhibitors. (c) Photolabeling of IFITM3 and PS1 by L505 in human brains (control: n=4, LOAD-L: n=5, LOAD-H: n=5). (d) Pearson’s correlation between γ-secretase activity and L505 labeled IFITM3 (Fig. 5c) in LOAD samples (n=10). (e) Double cross-linking of γ-secretase and IFITM3 by the dual probe, L631. WB reveals multiple protein complex species containing IFITM3, PS1-NTF, PS1-CTF, IFITM3 homodimer, IFITM3-PS1 and PS1-NTF-CTF heterodimers. (f) Schematic representation of the interaction between IFITM3 and γ-secretase, IFITM3 is near the active site and can be crosslinked with PS1-NTF. (g) IFITM3 connects infections and innate immunity with Aβ production and AD risk. (A) Pathogenic challenge, or other inflammatory conditions, induce the release of proinflammatory cytokines from astrocytes and microglia. (B) Cytokines upregulate IFITM3 expression in neurons and astrocytes that potentiates γ-secretase, increasing Aβ production. (C) As part of an innate immune response, Aβ acts as an antimicrobial or antiviral peptide. In turn, Aβ accumulation also triggers AD pathology. All WB images and graphs are representative of three independent experiments (except; c: 2 replicates).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Binding Assay, Control, Activity Assay, Labeling, Expressing

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: WT and Ifitm3 -/- mice were intranasally infected with ( a, b ) 1, 10, or 50 TCID50 of H5N1 avian influenza strain (2 independent experiments for doses 1 and 10 (n=10 mice) and 1 experiment for dose of 50 (n=5 mice)) or with ( c, d ) 1 or 10 TCID50 of H7N3 avian influenza strain (n=5 mice). a , c Viral titers from lung homogenates at day 3 post infection. b, d ELISA quantification of IL-6 levels in lung homogenates at day 3 post infection. a-d Error bars represent SEM. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. Only comparisons between WT and Ifitm3 -/- mice for each dose are shown.

Article Snippet: Membranes were blocked with 10% non-fat milk in Phosphate-buffered saline with 0.1% Tween-20 (PBST) and probed with antibodies against IFITM1 (Cell Signaling Technology, #13126), IFITM2 (Cell Signaling Technology, #13530), IFITM3 (ProteinTech, 11714-1-AP), and GAPDH (Thermo Scientific, ZG003).

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: ( a ) Schematic of in vitro infection with potentially zoonotic influenza viruses and representative example from infected A549 human lung cells. ( b ) The indicated A549 cells or THP-1 differentiated macrophages were treated +/- IFNβ for 18 hours, followed by infection with indicated viruses (MOI 1) for 24 hours. Percent infection was determined by flow cytometry and normalized to respective shControl or WT cells without IFNβ pre-treatment. Error bars represent SEM. P values are for the indicated comparisons and were determined by ANOVA followed by Tukey’s multiple comparisons test. Only statistical comparisons between shControl versus shIFITM3 and WT versus IFITM3 -/- are shown. Data are representative of 3 independent experiments each performed in triplicate (n=9). ( c, d ) Western blots of cell lysates at 18 hours +/- IFNβ treatment. Note that commercial IFITM3 antibodies weakly detect IFITM2 in addition to IFITM3. ( e ) The indicated A549 cells were infected with indicated viruses at a range of 0.001 to 10 MOI for 24 hours. Percent infection was determined by flow cytometry. Data are representative of 3 independent experiments each performed in triplicate (n=9). Error bars represent SEM. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. Only comparisons between control and knockdown cells are shown at each dose.

Article Snippet: Membranes were blocked with 10% non-fat milk in Phosphate-buffered saline with 0.1% Tween-20 (PBST) and probed with antibodies against IFITM1 (Cell Signaling Technology, #13126), IFITM2 (Cell Signaling Technology, #13530), IFITM3 (ProteinTech, 11714-1-AP), and GAPDH (Thermo Scientific, ZG003).

Techniques: In Vitro, Infection, Flow Cytometry, Western Blot, Control, Knockdown

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: HAP1 cells were treated +/- IFNβ for 18 hours, followed by infection with indicated viruses (MOI 1) for 24 hours. ( a ) Percent infection was determined by flow cytometry and normalized to results for WT cells without IFNβ pre-treatment. Error bars represent SEM. P values are for the indicated comparisons and were determined by ANOVA followed by Tukey’s multiple comparisons test. Only statistical comparisons between WT versus IFITM3 -/- are shown. Data are representative of 3 independent experiments each performed in triplicate (n=9). ( b ) Western blots of cell lysates at 18 hours +/- IFNβ treatment. ( c ) Example raw flow cytometry data for determination of normalized infection of WT and IFITM3 -/- HAP1 cells +/- IFNb treatment with each of the indicated virus strains as in ( a ). SSC, side scatter; Anti-influenza nucleoprotein antibody was detected in the APC channel with secondary antibody labeled with Alexafluor 647.

Article Snippet: Membranes were blocked with 10% non-fat milk in Phosphate-buffered saline with 0.1% Tween-20 (PBST) and probed with antibodies against IFITM1 (Cell Signaling Technology, #13126), IFITM2 (Cell Signaling Technology, #13530), IFITM3 (ProteinTech, 11714-1-AP), and GAPDH (Thermo Scientific, ZG003).

Techniques: Infection, Flow Cytometry, Western Blot, Virus, Labeling

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: HeLa cells were treated +/- IFNβ for 18 hours, followed by infection with indicated viruses (MOI 1) for 24 hours. ( a ) Percent infection was determined by flow cytometry and normalized to results for WT cells without IFNβ pre-treatment. Error bars represent SEM. P values are for the indicated comparisons and were determined by ANOVA followed by Tukey’s multiple comparisons test. Only statistical comparisons between WT versus IFITM3 -/- are shown. Data are representative of 3 independent experiments each performed in triplicate (n=9). ( b ) Western blots of cell lysates at 18 hours +/- IFNβ treatment. ( c ) Example raw flow cytometry data for determination of normalized infection of WT and IFITM1/2/3 -/- HeLa cells +/- IFNb treatment with each of the indicated virus strains as in a . SSC, side scatter; Anti-influenza nucleoprotein antibody was detected in the APC channel with secondary antibody labeled with Alexafluor 647.

Article Snippet: Membranes were blocked with 10% non-fat milk in Phosphate-buffered saline with 0.1% Tween-20 (PBST) and probed with antibodies against IFITM1 (Cell Signaling Technology, #13126), IFITM2 (Cell Signaling Technology, #13530), IFITM3 (ProteinTech, 11714-1-AP), and GAPDH (Thermo Scientific, ZG003).

Techniques: Infection, Flow Cytometry, Western Blot, Virus, Labeling

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: ( a ) Schematic of mouse passaging experiments. Initial intranasal infections were performed with 1,000 TCID50 of parental viruses. ( b ) Schematic of WT mouse challenge with parental or passaged viruses. ( c-h and j-l ) Groups of WT mice were challenged with equal doses of virus passaged 1, 5, or 10 times through WT or Ifitm3 -/- mice and compared to the parent virus (passage 0). ( c, e ) Viral titers from lung homogenates taken at day 7 ( c represents 2 independent experiments). Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s post hoc test. ( d, f ) ELISA quantification of IL-6 levels in lung homogenates of WT and IFITM3 KO mice at day 7 post infection ( d represents 2 independent experiments). Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. ( g, j ) Viral titers from lung homogenates taken at day 7 ( g ) or day 6 ( j ) post infection. Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s post hoc test. ( h, k ) ELISA quantification of IL-6 levels in lung homogenates of WT and IFITM3 KO mice at day 7 ( h ) or day 6 ( k ) post infection. Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. ( i, m ) Mutations found in the segments of A/California/04/2009 (H1N1) after serial passage through WT or Ifitm3 -/- mice. ( l ) Weight loss. Skull and crossbones indicate humane euthanasia of all animals infected with KO passage 10. Error bars represent SD of the mean, comparisons were made using the Mann-Whitney test (* P = 0.0022, ** P < 0.0001).

Article Snippet: Membranes were blocked with 10% non-fat milk in Phosphate-buffered saline with 0.1% Tween-20 (PBST) and probed with antibodies against IFITM1 (Cell Signaling Technology, #13126), IFITM2 (Cell Signaling Technology, #13530), IFITM3 (ProteinTech, 11714-1-AP), and GAPDH (Thermo Scientific, ZG003).

Techniques: Passaging, Virus, Enzyme-linked Immunosorbent Assay, Infection, MANN-WHITNEY

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: ( a ) Schematic of mouse passaging experiments. Initial intranasal infections were performed with 1,000 TCID50 of parental viruses. ( b ) Schematic of WT mouse challenge with parental or passaged viruses. ( c,d ) Groups of WT mice were challenged with equal doses of virus passaged 1, 5, or 10 times through WT or Stat1 -/- mice and compared to the parent virus (passage 0). ( c ) Viral titers from lung homogenates taken at day 7 post infection. Error bars represent SEM. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. ( d ) ELISA quantification of IL-6 levels in lung homogenates of WT and IFITM3 KO mice at day 7 post infection. Error bars represent SEM. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: Membranes were blocked with 10% non-fat milk in Phosphate-buffered saline with 0.1% Tween-20 (PBST) and probed with antibodies against IFITM1 (Cell Signaling Technology, #13126), IFITM2 (Cell Signaling Technology, #13530), IFITM3 (ProteinTech, 11714-1-AP), and GAPDH (Thermo Scientific, ZG003).

Techniques: Passaging, Virus, Infection, Enzyme-linked Immunosorbent Assay

Journal: Investigative and clinical urology

Article Title: IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

doi: 10.4111/icu.20230294

Figure Lengend Snippet: Fig. 1. Interferon-induced transmem- brane protein 3 (IFITM3) expression in the normal kidney and clear cell renal cell carcinoma (ccRCC). (A) In normal renal parenchyma, IFITM3 was immuno- reactive in glomerular parietal epithelial cells, capillaries (left), and damaged or atrophic tubules (right). IFITM3 ex- pression was inconspicuous in tubular epithelial cells (middle). (B) In ccRCC, IFITM3 was immunoreactive as a mem- branous pattern in low-grade tumors (left), whereas cytoplasmic patterns are observed in high-grade tumors (right) (magnification, ×400).

Article Snippet: For ectopic overexpression of IFITM3 proteins, a lentivirus containing the pCMV6-Entry vector cloned with IFITM3 ORF construct was purchased (RC201635, OriGene).

Techniques: Expressing

Journal: Investigative and clinical urology

Article Title: IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

doi: 10.4111/icu.20230294

Figure Lengend Snippet: Fig. 3. (A) The tumorigenicity of 786-O P/empty, 786-O suR/empty, 786-O P/IFITM3-overexpression, and 786-O suR/IFITM3-shRNA was evaluated in a xenograft model. Sunitinib was administered 31 days after transplantation. (B) Representative images 786-O P/IFITM3-overexpression (left upper), 786-O P/empty, 786-O suR/IFITM3-shRNA, and 786-O suR/empty were presented clockwise. Representative images of the high-power field were presented as inset. IFITM3, interferon-induced transmembrane protein 3 (magnification, ×40; ×400).

Article Snippet: For ectopic overexpression of IFITM3 proteins, a lentivirus containing the pCMV6-Entry vector cloned with IFITM3 ORF construct was purchased (RC201635, OriGene).

Techniques: Over Expression, shRNA, Transplantation Assay

Journal: Investigative and clinical urology

Article Title: IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

doi: 10.4111/icu.20230294

Figure Lengend Snippet: Fig. 5. The interferon-induced trans- membrane protein 3 (IFITM3)-induced acquired tyrosine kinase inhibitor (TKI) resistance mechanism represented in a schematic diagram. In patients resistant to sunitinib, overexpression of IFITM3 is observed. The binding of IFITM3 to TRAF6 leads to the activation of TRAF6 associated with IRAK1, which induces oncogenic signaling pathways, includ- ing MAPK/AP-1 pathways. As a result, clear cell renal cell carcinoma (ccRCC) can accelerate tumor growth, survival, and proliferation despite TKI treatment.

Article Snippet: For ectopic overexpression of IFITM3 proteins, a lentivirus containing the pCMV6-Entry vector cloned with IFITM3 ORF construct was purchased (RC201635, OriGene).

Techniques: Membrane, Over Expression, Binding Assay, Activation Assay, Protein-Protein interactions

Journal: EMBO Reports

Article Title: Inhibition of IRGM establishes a robust antiviral immune state to restrict pathogenic viruses

doi: 10.15252/embr.202152948

Figure Lengend Snippet: RNA isolated from control and IRGM knockdown HT‐29 cells and subjected to qRT–PCR with indicated viral restriction factor genes ( n = 3, mean ± SD, ** P < 0.005, *** P < 0.0005, Student’s unpaired t ‐test). The graph depicts the knockdown efficiency of control and IRGM shRNA stable HT‐29 cells ( n = 3, mean ± SD, **** P < 0.00005, Student’s unpaired t ‐test). The graph depicts the IFN‐β levels in serum of Irgm1 wild‐type and KO mice ( n = 3 mice each group, mean ± SD, **** P < 0.00005, Student’s unpaired t ‐test). The qRT–PCR analysis of APOBEC3G, OAS1, ISG15, MX1 and IFITM3 with RNA isolated from control or Flag IRGM overexpressing HT‐29 cells ( n = 3, Mean ± SD, * P < 0.05, ** P < 0.005, *** P < 0.0005, Student's unpaired t ‐test). The qRT–PCR analysis of OAS1, ISG15 and MX1 with RNA isolated from control or IRGM shRNA or Flag IRGM complemented IRGM shRNA HT‐29 cells. n = 3, Mean ± SD, * P < 0.05, ** P < 0.005, *** P < 0.0005, Student's unpaired t ‐test. Pictorial representation of sequential events of antigen uptake, processing, and presentation via Class I and Class II MHC Pathways. Created using Biorender.com. The graph depicts the knockdown efficiency upon transfection of control and si‐IRGM in THP‐1 cells ( n = 3, mean ± SD, *** P < 0.0005, Student’s unpaired t ‐test). Transferrin uptake assay shown by representative confocal images and flow cytometry analysis of control and si‐IRGM transfected THP‐1 cells treated with AF488 Transferrin (green) (10 μg/ml, 30 min). Graph depicts the mean fluorescence intensity of transferrin uptake in control and si‐IRGM transfected THP‐1 cells treated with AF488 transferrin. Scale, 5 μm (upper panel); Scale, 3 μm, (lower panel). Representative confocal images of H‐2Kb‐SIINFEKL (red) in Irgm1 +/+ and Irgm1 −/− BMDMs treated with OVA (2 mg/ml, 3 h). Scale, 25 μm.

Article Snippet: Primary antibodies used in Western blotting with dilutions: Actin (Abcam #ab6276; 1:5,000), IRGM antibody rodent specific (CST #14979; 1:1,000), IRGM (Abcam #ab69494; 1:500), MX1 (CST #37849; 1:1,000), OAS1 (CST #14498; 1:1,000), ISG15 (CST #2743; Santacruz sc‐166755; 1:1,000; 1:1,000), SAMHD1 (CST #12361; 1:1,000), BST2 (CST #19277; 1:1,000), viperin (CST #13996; 1:1,000), APOBEC3G (CST #43584; 1:1,000), IFITM3 (CST #59212; 1:1,000), PKR B10 (sc‐6282; 1:1,000), p‐PKR (#ab32036; 1:1,000), EIF2 Alpha (CST #9722; 1:1,000), p‐EIF2 Alpha (CST #3398S; 1:1,000), G3BP (BD Bioscience #611126; 1:1,000), GAPDH (CST #2118; 1:1,000), ACE2 (R&D systems #AF933; 1:1,000), HRP‐conjugated secondary antibodies were purchased from Santa Cruz (1:2,000) or Promega (1:5,000) or Abcam (1:10,000) or Novus (1:5,000).

Techniques: Isolation, Control, Knockdown, Quantitative RT-PCR, shRNA, Transfection, Flow Cytometry, Fluorescence

Journal: Virologica Sinica

Article Title: Elevated interferon-induced transmembrane protein 3 in platelets and megakaryocytes suppresses Crimean-Congo hemorrhagic fever viral infection by interacting with glycoprotein Gc

doi: 10.1016/j.virs.2025.05.002

Figure Lengend Snippet: Identification of differentially expressed genes in platelets of CCHFV-infected mice. Three groups of C57BL/6J mice (6–8 weeks old, female, n ​= ​10/group) were inoculated with CCHFV (1.56 ​× ​10 5 TCID 50 /mouse) intraperitoneally, and two mock groups (n ​= ​10/group) received brain homogenate in the same route and served as control groups. On day 3 post-inoculation, mice were euthanized and blood was collected. Platelets were then purified and applied to transcriptome sequencing. A Hierarchical clustering of global gene expression data revealed that samples from the CCHFV-infected group (cyan) were closely clustered, and were distinct from those of the healthy group (pink). The data are presented by the Pearson correlation coefficient (Pearson's r) between different groups. B KEGG annotation and expression analysis of the genome. The ten pathways with the smallest adjusted P -values ( p.adjust ) are shown. Pathways were sorted according to gene ratio. C GO enrichment analysis highlighting representative functional categories enriched among differentially expressed genes (DEGs) in CCHFV-infected mice ( P ​< ​0.01). The ten entries with the smallest p.adjust from the three GO categories (CC: Cell Component; MF: Molecular Function; BP: Biological Process) are shown. D Volcano plot showing the differential expression of genes (DEGs) in platelets of CCHFV-infected mice in comparison to healthy controls. Red dots represent upregulated genes, blue dots indicate downregulated genes, and grey dots show genes with no significant differences. The top five genes exhibiting increased transcription levels the most significantly more than the other genes are labelled. E The heatmap exhibited the top 20 DEGs with a significantly increased or decreased transcription levels. The data has been normalized by row and is displayed as a z-score. F Validation of increased IFITM3 protein expression in platelets of infected mice via Western blot analysis, with GAPDH as the internal control. Three other groups of mice (n ​= ​10/group) were infected with CCHFV (Infection) in the same route as described above. Platelets were purified on day 3 post-isolation. After confirming platelet purity >97%, IFITM3 expression was examined by Western blot. Platelets from three groups of healthy mice inoculated with culture medium (Mock) were used as control, and GAPDH expression was blotted as inner control of platelets.

Article Snippet: The rabbit anti-human IFITM3 antibody ([KO Validated] IFITM3 Rabbit pAb (A13070)), which is capable to recognize both human and mouse IFITM3, was purchased from ABclonal Technology, Inc (Wuhan, China).

Techniques: Infection, Control, Purification, Sequencing, Gene Expression, Expressing, Functional Assay, Quantitative Proteomics, Comparison, Biomarker Discovery, Western Blot, Isolation

Journal: Virologica Sinica

Article Title: Elevated interferon-induced transmembrane protein 3 in platelets and megakaryocytes suppresses Crimean-Congo hemorrhagic fever viral infection by interacting with glycoprotein Gc

doi: 10.1016/j.virs.2025.05.002

Figure Lengend Snippet: Characterization of the properties of CCHFV infection in MEG-01 and HEK293 Cells. MEG-01 and HEK293 ​cells seeded in 24-well plates were incubated with CCHFV (MOI ​= ​5) for 2 ​h. After infection, cells were washed three times with PBS and cultured at 37 ​°C in a 5% CO 2 incubator. Cells were collected at indicated time points for subsequent analyses. A The CCHFV infection efficiencies in MEG-01 and HEK293 ​cells collected at 48 ​h p.i. were analyzed by flow cytometry using CCHFV NP antibody. B CCHFV NP and IFITM3 expression were examined by Western blotting in MEG-01 and HEK293 ​cells collected at indicated time points post CCHFV infection, with β-actin as the inner control. C The transcription levels of ifitm3 in MEG-01 and HEK293 ​cells were examined by qRT-PCR at indicated time points post CCHFV infection, and were normalized to GAPDH. D Quantitative analysis of the viral copies by qRT-PCR in the culture supernatant (left) and in the MEG-01 and HEK293 ​cells (right), respectively. E Visualization of CCHFV infection in MEG-01 and HEK293 ​cells by IFAs. IFITM3 and CCHFV NP expression was blotted in cells infected with CCHFV at 48 ​h p.i. or with mock infection. Cells were immunostained using antibodies against CCHFV NP (red) and IFITM3 (green), respectively. Nuclei were stained with Hoechst33258 (blue). Scale bar, 50 ​μm. Differences between the two groups were analyzed using One-way ANOVA to calculate the P -value to evaluate the differences in expression levels at different time points in the same group. P ​< ​0.05 was considered statistically significant.

Article Snippet: The rabbit anti-human IFITM3 antibody ([KO Validated] IFITM3 Rabbit pAb (A13070)), which is capable to recognize both human and mouse IFITM3, was purchased from ABclonal Technology, Inc (Wuhan, China).

Techniques: Infection, Incubation, Cell Culture, Flow Cytometry, Expressing, Western Blot, Control, Quantitative RT-PCR, Staining

Journal: Virologica Sinica

Article Title: Elevated interferon-induced transmembrane protein 3 in platelets and megakaryocytes suppresses Crimean-Congo hemorrhagic fever viral infection by interacting with glycoprotein Gc

doi: 10.1016/j.virs.2025.05.002

Figure Lengend Snippet: Overexpression of IFITM3 inhibits CCHFV infection in MEG-01 (A – C) and HEK293 (D – F) Cells. Cells were infected with CCHFV (MOI=5) and collected at indicated time points for subsequent analyses. A Western blot analyses of CCHFV NP and IFITM3 expression in MEG-01 (WT) and MEG-01- hifitm3 -OE (IFITM3-OE) cells at indicated time points, with β-actin as the internal control. B Quantitative RT-PCR to determine the RNA loads of CCHFV S segment in both supernatants (left) and cells (right) at indicated time points. C Confocal microscopy images showing CCHFV NP and IFITM3 expression and subcellular localization. Cells are immunostained using antibodies against CCHFV NP (green) and IFITM3 (red), respectively. D Western blot analyses of CCHFV NP and IFITM3 expression in HEK293 (WT) and HEK293- hifitm3 -OE (IFITM3-OE) cells at indicated time points, with β-actin as the internal control. E Quantitative RT-PCR to determine the RNA loads of CCHFV S segment in both supernatants (left) and cells (right) at indicated time points. F Confocal microscopy images showing CCHFV NP and IFITM3 expression and subcellular localization. Cells were immunostained using antibodies against CCHFV NP (green) and IFITM3 (red), respectively. Nuclei were stained with Hoechst33258 (blue). Images were taken using a Leica confocal microscope. Scale bar, 20 ​μm. The data between groups were analyzed using two-way ANOVA. Data are expressed as the mean ​± ​SD. P ​< ​0.05 indicates a statistically significant difference, ∗ P ​< ​0.05, ∗∗ P ​< ​0.01, ∗∗∗∗ P ​< ​0.0001.

Article Snippet: The rabbit anti-human IFITM3 antibody ([KO Validated] IFITM3 Rabbit pAb (A13070)), which is capable to recognize both human and mouse IFITM3, was purchased from ABclonal Technology, Inc (Wuhan, China).

Techniques: Over Expression, Infection, Western Blot, Expressing, Control, Quantitative RT-PCR, Confocal Microscopy, Staining, Microscopy

Journal: Virologica Sinica

Article Title: Elevated interferon-induced transmembrane protein 3 in platelets and megakaryocytes suppresses Crimean-Congo hemorrhagic fever viral infection by interacting with glycoprotein Gc

doi: 10.1016/j.virs.2025.05.002

Figure Lengend Snippet: Knockout IFITM3 promotes CCHFV infection in MEG-01 (A – C) and HeLa (D – F) cells. Cells were infected with CCHFV (MOI ​= ​5), and collected at indicated time points for subsequent analyses. A Western blot analyses of CCHFV NP and IFITM3 expression in MEG-01 (WT) and MEG-01- hifitm3 -KO (IFITM3-KO) cells at indicated time points, with β-actin as the internal control. B Quantitative RT-PCR to determine the RNA loads of CCHFV S segment in both supernatants (left) and cells (right) at indicated time points. C Confocal microscopy images showing CCHFV NP and IFITM3 expression and subcellular localization. Cells are immunostained using antibodies against CCHFV NP (green) and IFITM3 (red), respectively. D Western blot analyses of CCHFV NP and IFITM3 expression in HeLa (WT) and HeLa- hifitm3 -KO (IFITM3-KO) cells at indicated time points, with β-actin as the internal control. E Quantitative RT-PCR to determine the RNA loads of CCHFV S segment in both supernatants (left) and cells (right) at indicated time points. F Confocal microscopy images showing CCHFV NP and IFITM3 expression and subcellular localization. Cells were immunostained using antibodies against CCHFV NP (green) and IFITM3 (red), respectively, and nuclei were stained with Hoechst33258 (blue). Images were taken using a Leica confocal microscope. Scale bar, 20 ​μm. The data between groups were analyzed using two-way ANOVA. Data are expressed as the mean ​± ​SD. P ​< ​0.05 indicates a statistically significant difference, ∗ P ​< ​0.05, ∗∗ P ​< ​0.01, ∗∗∗∗ P ​< ​0.0001.

Article Snippet: The rabbit anti-human IFITM3 antibody ([KO Validated] IFITM3 Rabbit pAb (A13070)), which is capable to recognize both human and mouse IFITM3, was purchased from ABclonal Technology, Inc (Wuhan, China).

Techniques: Knock-Out, Infection, Western Blot, Expressing, Control, Quantitative RT-PCR, Confocal Microscopy, Staining, Microscopy

Journal: Virologica Sinica

Article Title: Elevated interferon-induced transmembrane protein 3 in platelets and megakaryocytes suppresses Crimean-Congo hemorrhagic fever viral infection by interacting with glycoprotein Gc

doi: 10.1016/j.virs.2025.05.002

Figure Lengend Snippet: IFITM3 interacts with CCHFV Gc protein. HEK293T cells were transfected with pEGFP-N1- hifitm3 (IFITM3) or pEGFP-N1 (Vector) for 24 ​h and then infected with CCHFV (MOI ​= ​5) for 48 ​h. After lysis, the cells were pulled down with the beads against the enhanced green fluorescent protein (anti-EGFP). A Western blot analyses of CCHFV Gc, NP, and Gn, EGFP, and β-actin in the cell lysates and outputs after pulling down using anti-EGFP. B Confocal microscopy images showing the viral protein (Gc) and IFITM3 (EGFP) are co-localized. Images were taken using a Leica confocal microscope. Scale bar, 20 ​μm. C Quantification of CCHFV (MOI ​= ​10) binding (left) and internalization (right) to MEG-01 (WT) and MEG-01- hifitm3- OE (IFITM3-OE) cells. For binding assays, cells were incubated with CCHFV on ice for 1 ​h and were washed three times with ice-cold PBS to remove unbound viruses. Cells were then harvested to determine CCHFV S RNA by qRT-PCR. For internalization assays, cells were incubated with CCHFV at 4 ​°C for 1 ​h, then moved to 37 ​°C and incubated for 1h to allow viral internalization. Cells were washed with PBS containing 500 ​ng/mL proteinase K to remove the uninternalized virus. Internalized viruses were quantified by qRT-PCR. GAPDH was blotted as an internal control. The CCHFV RNA loads were normalized to MEG-01 ​cells using 2 −ΔΔCt method. The significant difference was determined using the student's t-test . P ​< ​0.05 indicates a statistically significant difference, ∗∗ P ​< ​0.01, ∗∗∗ P ​< ​0.001.

Article Snippet: The rabbit anti-human IFITM3 antibody ([KO Validated] IFITM3 Rabbit pAb (A13070)), which is capable to recognize both human and mouse IFITM3, was purchased from ABclonal Technology, Inc (Wuhan, China).

Techniques: Transfection, Plasmid Preparation, Infection, Lysis, Western Blot, Confocal Microscopy, Microscopy, Binding Assay, Incubation, Quantitative RT-PCR, Virus, Control

Journal: Virologica Sinica

Article Title: Elevated interferon-induced transmembrane protein 3 in platelets and megakaryocytes suppresses Crimean-Congo hemorrhagic fever viral infection by interacting with glycoprotein Gc

doi: 10.1016/j.virs.2025.05.002

Figure Lengend Snippet: CIL-TMD binds to the CCHFV Gc. HEK293T cells were co-transfected with phMCV-Gc-S-tag together with the pEGFP-N1 plasmid expressing IFITM3 or the truncated IFITM3 fragments or the pEGFP-N1 plasmid as control (Vector). At 24 ​h post transfection, cells were collected and lysed using an IP buffer. A and D The interaction of IFITM3 and its truncated fragments with CCHFV Gc were analyzed via S-pulldown assays followed by Western blotting, with β-actin as the internal control. B Schematic diagram of the constructs of truncated IFITM3 used in this study, each fused with EGFP at its N terminus. C and E Visualization of IFITM3 or its truncations (green) and CCHFV Gc (red) in cells using confocal microscopy. The transfected cells were fixed and permeabilized, and then incubated with S-tag antibody by IFAs. Nuclei were stained with Hoechst 33,258. Scale bar, 10 ​μm. F Schematic diagram of the 3D structures of IFITM3 and CCHFV Gc, which were predicted using Alpha fold 3. The IFITM3 domains and the regions and amino acids which could be involved in the interaction are indicated.

Article Snippet: The rabbit anti-human IFITM3 antibody ([KO Validated] IFITM3 Rabbit pAb (A13070)), which is capable to recognize both human and mouse IFITM3, was purchased from ABclonal Technology, Inc (Wuhan, China).

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Western Blot, Construct, Confocal Microscopy, Incubation, Staining