human his ifitm1 recombinant bait proteins  (Proteintech)

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Porcine IFITM1 enhances PEDV infection in LLC-PK1 cells. ( A ) Western blot analysis of porcine IFITM1 and IFITM2/3 overexpression in LLC-PK1 cells transduced with lentiviral vectors expressing porcine HA-tagged porcine IFITM1, porcine IFITM2/3, or an empty vector as a negative control. Porcine IFITM1- or IFITM2/3-overexpressing LLC-PK1 cells were infected with the PEDV-HM strain. ( B ) Viral RNA copies in the supernatant were quantified by RT-qPCR and are presented as copy numbers. The data are presented as the means ± s.d. from three technical repeats. ( C ) Viral titers in the supernatant were measured via the TCID 50 assay. The data are presented as the means ± s.d. from three independent experiments. ( D ) LLC-PK1 cells overexpressing porcine IFITM1 or IFITM2/3 were infected with rPEDV-HM-EGFP (0.1 MOI), and the EGFP-fluorescent cells were imaged via fluorescence microscopy at 24 hpi. The green cells represent PEDV-infected cells, while background cells were visualized via white light. Scale bar: 200 µm.

Article Snippet: A monoclonal antibody for human IFITM1 (cat#: 60074-1-lg) was obtained from Proteintech (Shanghai, China).

Techniques: Infection, Western Blot, Over Expression, Transduction, Expressing, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Fluorescence, Microscopy

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Interaction and colocalization of the PEDV S protein with IFITM proteins. ( A ) Co-IP assays were performed to assess the interaction between the PEDV S1 protein and human or porcine IFITM proteins. HEK293T cells were cotransfected with plasmids encoding PEDV S1-Fc and various HA-tagged IFITM proteins. At 24 h post-transfection, the cells were harvested. Immunoprecipitation was conducted using an Fc tag antibody, and the presence of IFITM proteins was detected by Western blotting (IB: Anti-HA). Whole-cell lysates (WCLs) were analyzed to confirm expression levels (IB: Anti-Fc, Anti-HA, Anti-β-Tubulin). The Co-IP was repeated three times, yielding similar results. ( B ) Confocal microscopy images showing the intracellular localization of porcine IFITM1 in LLC-PK1 cells are presented. IFITM1 (red) colocalizes with clathrin (green), EEA1 (green), Rab7 (green), and LAMP7 (green), as indicated in the merged images. DAPI (blue) was used to stain the nuclei. ( C ) Confocal microscopy images demonstrating the colocalization of the PEDV S protein (red) with HA-IFITM1 (green) in LLC-PK1 cells are shown. The merged image shows the nuclei stained with DAPI (blue).

Article Snippet: A monoclonal antibody for human IFITM1 (cat#: 60074-1-lg) was obtained from Proteintech (Shanghai, China).

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Expressing, Confocal Microscopy, Staining

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Porcine IFITM1 enhanced PEDV infection in porcine small intestinal organoids. ( A ) Porcine small intestinal crypts were isolated from neonatal piglets and cultured in vitro . A representative image depicting the morphology of porcine small intestinal organoids is shown. Scale bar: 200 µm. ( B ) Immunofluorescence confirmation of HA-tagged porcine IFITM1 overexpression in PSIOs. Representative images displaying IFITM1 (HA, green) and nuclei (DAPI, blue) are presented. ( C ) The expression of IFITM1 was verified via immunofluorescence in a 2D organoid culture system. Representative images showing HA (green) and nuclei (DAPI, blue) staining are presented. ( D ) Analysis of viral infection in IFITM1-overexpressing and control 2D organoids following PEDV infection is shown. IFITM1-overexpressing or control 2D organoids were infected with PEDV-HM (0.1 MOI). At 24 hpi, the virus titer in the supernatant was quantified according to the TCID 50 . The data are presented as the means ± s.d. from three independent experiments. **, P < 0.01. ( E ) IFITM1-overexpressing or control 2D organoids were infected with rPEDV-HM-EGFP (0.1 MOI), and the EGFP-fluorescent cells were imaged via fluorescence microscopy at 24 hpi. The green cells represent PEDV-infected cells, and the background cells were visualized under white light. Scale bar: 200 µm.

Article Snippet: A monoclonal antibody for human IFITM1 (cat#: 60074-1-lg) was obtained from Proteintech (Shanghai, China).

Techniques: Infection, Isolation, Cell Culture, In Vitro, Immunofluorescence, Over Expression, Expressing, Staining, Control, Virus, Fluorescence, Microscopy

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: Vero (A), 293 (B), and HeLa (C) cells were incubated with the indicated concentrations of IFN-α for 24 h and then transfected with AiV replicon RNA. At the indicated time points after transfection, cell lysates were harvested, and luciferase activity was measured. Each right panel represents the protein levels of IFITM1, IFITM2, or IFITM3 by immunoblotting for cell lysates harvested from experiments shown in the left panels. Data are the means ±SD of at least three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Incubation, Transfection, Luciferase, Activity Assay, Western Blot

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A–C) 293 (A), HeLa (B), or Vero (C) cell lines stably expressing tetracycline (Tet)-inducible IFITM1, IFITM2 or IFITM3 were cultured with or without Tet for 72 h and then transfected with AiV replicon RNA. Cell lysates were harvested at the indicated time points after transfection and then assayed for luciferase activity. The peak activity obtained for cells in the absence of Tet was taken as 100%. Tet-induced IFITM1, IFITM2, or IFITM3 protein expression in each cell line was detected by Western blotting (bottom panels of (A) and right panel of (B) or right panel of (C)). (D) HeLa cells were transfected with the control, IFITM1, IFITM2, or IFITM3 siRNA for 72 h and then transfected with replicon RNA. At the indicated time points after replicon RNA transfection, cell lysates were harvested and subjected to the luciferase assay. The maximum value obtained for cells treated with control siRNA was taken as 100%. (E) IFITM1, IFITM2, or IFITM3 knockdown was confirmed by Western blotting. (F) Cell viability was determined by the CellTiter-Glo assay. Data are the means ± SD of at least three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Stable Transfection, Expressing, Cell Culture, Transfection, Luciferase, Activity Assay, Western Blot, Control, Knockdown, Glo Assay

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: Vero (A), 293 (B), and HeLa (C) cells were incubated with the indicated concentrations of IFN-α for 24 h and then transfected with AiV replicon RNA. At the indicated time points after transfection, cell lysates were harvested, and luciferase activity was measured. Each right panel represents the protein levels of IFITM1, IFITM2, or IFITM3 by immunoblotting for cell lysates harvested from experiments shown in the left panels. Data are the means ±SD of at least three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Incubation, Transfection, Luciferase, Activity Assay, Western Blot

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A–C) 293 (A), HeLa (B), or Vero (C) cell lines stably expressing tetracycline (Tet)-inducible IFITM1, IFITM2 or IFITM3 were cultured with or without Tet for 72 h and then transfected with AiV replicon RNA. Cell lysates were harvested at the indicated time points after transfection and then assayed for luciferase activity. The peak activity obtained for cells in the absence of Tet was taken as 100%. Tet-induced IFITM1, IFITM2, or IFITM3 protein expression in each cell line was detected by Western blotting (bottom panels of (A) and right panel of (B) or right panel of (C)). (D) HeLa cells were transfected with the control, IFITM1, IFITM2, or IFITM3 siRNA for 72 h and then transfected with replicon RNA. At the indicated time points after replicon RNA transfection, cell lysates were harvested and subjected to the luciferase assay. The maximum value obtained for cells treated with control siRNA was taken as 100%. (E) IFITM1, IFITM2, or IFITM3 knockdown was confirmed by Western blotting. (F) Cell viability was determined by the CellTiter-Glo assay. Data are the means ± SD of at least three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Stable Transfection, Expressing, Cell Culture, Transfection, Luciferase, Activity Assay, Western Blot, Control, Knockdown, Glo Assay

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A) A mammalian two-hybrid assay was performed by transfecting Vero cells with the indicated combinations of pACT and pBIND plasmids along with the pG5luc firefly luciferase reporter plasmid. At 48 h after transfection, the normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) was measured and represented as fold of the control activity, which was obtained by combination with the empty pACT or empty pBIND plasmid. Data are the mean ± SD of at least three independent experiments. (B) 293T cells were transfected for 24 h with FLAG-tagged IFITM1 and HA-tagged 3Cm, 2B, 2BC, 2C, 3A, or 3AB, or HA-tagged IFITM1 and FLAG-tagged TGN46 expression plasmids, as indicated, followed by coimmunoprecipitation (IP) with an anti-FLAG, -HA or control IgG antibody. The resulting immunoprecipitates and whole-cell lysates were subjected to immunoblotting (IB) with anti-FLAG and anti-HA antibodies. (C) Vero cells were transfected with FLAG-IFITM1. At 24 h after post-transfection, the cells were fixed and double stained with anti-IFITM and anti-EEA1, anti-CD63, or anti-LBPA antibodies, as indicated. Pearson correlation coefficient analyses for data were obtained from ≥10 cells. Correlation coefficients are presented as the mean and standard deviation. (D) Vero cells was transfected with FLAG-IFITM1 and HA-2B, HA-2BC, HA-2C, HA-3A, or HA-3AB. At 24 h after transfection, cells were fixed and stained with anti-FLAG and anti-HA antibodies. Bars, 4 μm. Pearson correlation coefficient analyses for data were obtained from 4–8 cells.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Two Hybrid Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Control, Expressing, Western Blot, Staining, Standard Deviation

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A) A mammalian two-hybrid analysis was carried out to examine interactions between IFITM1 and ACBD3, PI4KB, OSBP, or CERT, and the results are shown as described in . Data are the mean ± SD of at least three independent experiments. (B) HA-tagged IFITM1 and FLAG-tagged ACBD3, PI4KB, OSBP, or CERT were cotransfected into 293T cells, and the cell lysates were subjected to immunoprecipitation with anti-HA antibody or control IgG. The resulting immunocomplexes and whole-cell lysates were detected by anti-FLAG and anti-HA antibodies. (C) Vero cells were transfected with FLAG-IFITM1. At 24 h, the cells were labeled with anti-FLAG and anti-ACBD3 (top), anti-PI4KB (middle), or anti-OSBP (bottom) antibodies. (D) Vero IFITM1 cells were incubated with Tet (−) or Tet (+) for 72 h, and the cells were fixed and double stained with the indicated antibodies. (E) Vero cells were transfected with HA-IFITM1. At 24 h, the cells were labeled using anti-HA, anti-OSBP and anti-EEA1, or anti-CD63 antibodies. Bars, 4 μm. Pearson correlation coefficient analyses for data were obtained from ≥10 cells. Correlation coefficients are presented as the mean and standard deviation (C-E).

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Immunoprecipitation, Control, Transfection, Labeling, Incubation, Staining, Standard Deviation

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A–F) Vero cells were pretreated with DMSO (as the control) or the indicated concentrations of 25-HC (A), U18666A (B), or BF738735 (C) for 24 h and then transfected with AiV replicon RNA. At 10 h after transfection, the cells were analyzed for luciferase activity. Data were normalized to the DMSO treatment, and cell viability was scored. (D–F) Vero IFITM1 cells were incubated with or without Tet for 48 h and then treated with mock (–) or 1 μM of 25-HC (D), 1.5 μM of U18666A (E), or 150 nM of BF738735 (F). After 24 h, the cells were transfected with AiV replicon RNA, and luciferase activity was determined at 10 h after transfection. (G) Cell viability was measured in parallel. The maximum value obtained for Tet (–) drug-untreated cells was taken as 100%. Data are the mean ± SD of at least three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Control, Transfection, Luciferase, Activity Assay, Incubation

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A–E) Vero-IFITM1 cells were incubated with or without Tet for 48 h and then treated with mock (A and D), 1 μM 25-HC (B), 1.5 μM U18666A (C), or 150 nM BF738735 (E), followed by pCMV-polyprotein transfection. At 24 h after transfection, the cells were fixed and stained with filipin III (A–C) or anti-PI4P (D and E), anti-IFITM1 and anti-2B antibodies. Bars, 4 μm. Pearson’s correlation coefficient was measured for individual cells.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Incubation, Transfection, Staining

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A and B) Vero-IFITM1 cells were incubated with or without Tet for 48 h, and then treated with 3 μM AY9944. After 24 h, the cells were fixed and stained with filipin III and the indicated antibodies. Bars, 4 μm. Pearson correlation coefficient analyses for data were obtained from ≥10 cells. Correlation coefficients are presented as the mean and standard deviation.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Incubation, Staining, Standard Deviation