rabbit anti ifitm2  (Proteintech)

Journal: Intractable & Rare Diseases Research

Article Title: The novel role of IFITM1-3 in myogenic differentiation of C2C12 cells

doi: 10.5582/irdr.2023.01050

Figure Lengend Snippet: Primer sequences for RT-qPCR

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature, and then the membranes were incubated with primary antibodies, namely, rabbit anti-desmin (ab32362, Abcam, USA), rabbit anti-MyoD (ab203383, Abcam, USA), rabbit anti-MYF5 (ab125301, Abcam, USA), rabbit anti-IFITM1 (bs-1031R, Bioss, China), rabbit anti-IFITM2 (bs-15517R, Bioss, China), mouse anti-IFITM3 (bsm-51629M, Bioss, China), and rabbit anti-GAPDH (ab8245, Abcam, USA), at 4°C overnight.

Techniques: Sequencing

Journal: iScience

Article Title: IFITM1 inhibits trophoblast invasion and is induced in placentas associated with IFN-mediated pregnancy diseases

doi: 10.1016/j.isci.2023.107147

Figure Lengend Snippet: Upregulation of IFITMs in HTR8/SVneo cells following IFN-β treatment was evaluated by western blot and immunofluorescence IFN-β inhibits invasion of HTR8/SVneo cells (A–E) and primary human EVCTs (F–I). HTR8/SVneo cells and EVCTs were incubated for 48 h with IFN-β (10, 100, or 1000 IU/mL). (A) Cell lysates (40 μg) were loaded in 4–15% SDS-PAGE gradient gels and blots were probed for IFITM1, IFITM2/3, and vinculin expression. One representative experiment (out of 3) is shown. (B) Representative image of HTR8/SVneo cells immunostained for cytokeratin 7 (marker of trophoblast, magenta) and IFITM1 (green) or IFITM2/3 (red), and then counterstained with DAPI (blue). Scale bars: 10 μm. Inhibition of HTR8/SVneo cell invasion following IFN-β treatment , quantified using IncuCyte technology (C–E) . HTR8/SVneo cells were cultured in a monolayer in 96-well plates. Cells were treated for 48 h, then a wound was made using a 96-well wound maker; this was covered by Matrigel and cells were imaged with Incucyte every 30 min for 48 h. (C) Representative images of HTR8/SVneo cell invasion at 48 h. The blue region denotes the area of the initial wound (light blue line) covered by advancing cells. (D) Time course of wound closure expressed as relative wound density (%). Values are reported as mean ± SEM. (E) Comparison between vehicle and IFN-β-treated cells was performed using an analysis of the area under the curve of the replicates represented in (D). Values are reported as mean + SEM (n = 6 wells/condition, n = 3 experiments). Statistical analysis was performed with one-way ANOVA to compare treatment to vehicle controls. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Upregulation of IFITMs in EVCTs following IFN-β treatment was evaluated by western blot (F) and immunofluorescence (G) . (F) Cell lysates (40 μg) from isolated EVCTs were loaded in 4–15% SDS-PAGE gradient gels and blots were probed for IFITM1, IFITM2/3, and vinculin expression. One representative experiment (out of 3) is shown. (G) Representative image of isolated EVCTs and placental explants immunostained for cytokeratin 7 (marker of trophoblast, magenta) and IFITM1 (green) or IFITM2/3 (red), and then counterstained with DAPI (blue). Scale bars: 10 μm. Inhibition of EVCT invasion following IFN-β treatment using villous explant culture model (H–I) . (H) Representative image of extravillous explants cultured on Matrigel and immunostained for integrin α5 (ITGA5, marker of invasion, red). Scale bars: 100 μm. (I) Statistical analysis of the invasion distance of ITGA5 + EVCTs, as representatively shown in H. Values are presented as mean + SD (n = 3 explants per condition, n = 3 placentas). One-way ANOVA was performed to compare treatment to vehicle control; ∗∗∗∗p < 0.0001.

Article Snippet: Rabbit anti-Fragilis [IFITM2/3] Monoclonal antibody (clone EPR5242) , Abcam , Cat# ab109429; RRID: AB_10865792.

Techniques: Western Blot, Immunofluorescence, Incubation, SDS Page, Expressing, Marker, Inhibition, Cell Culture, Isolation

Journal: iScience

Article Title: IFITM1 inhibits trophoblast invasion and is induced in placentas associated with IFN-mediated pregnancy diseases

doi: 10.1016/j.isci.2023.107147

Figure Lengend Snippet: Ectopic expression of FLAG and IFITMs in HTR8/SVneo cells transduced with IFITM1–3 was verified by western blot, immunofluorescence, and flow cytometry IFITM1 inhibits invasion of HTR8/SVneo cells (A–E) and primary human EVCTs. (A) Cell lysates (40 μg) were loaded in 4–15% SDS-PAGE gradient gels and blots were probed for IFITM1, IFITM2/3, and vinculin expression. A representative experiment is shown. (B) Representative image of transduced HTR8/SVneo cells immunostained for FLAG (yellow) and IFITM1 (green) or IFITM2/3 (red), and then counterstained with DAPI (blue). Scale bar: 10 μm. (C) HTR8/SVneo cells transduced with a vector containing IFITM1 (green), IFITM2 (orange), IFITM3 (magenta), or a control empty vector (dark blue) were stained with antibodies for IFITM1, IFITM2/3, and FLAG, as well as corresponding isotypes. Cells were then analyzed by flow cytometry. Inhibition of IFITM1-transduced HTR8/SVneo cell invasion , quantified using IncuCyte technology (D–F) . HTR8/SVneo cells transduced with IFITM1–3 were cultured in a monolayer in 96-well plates and a wound was made using a 96-well wound maker; this was covered by Matrigel and cells were imaged with Incucyte every 30 min for 48 h. (D) Representative images of transduced HTR8/SVneo cell invasion at 48 h. The blue region denotes the area of the initial wound (light blue line) covered by advancing cells. (E) Time course of wound closure expressed as relative wound density (%). Values are reported as mean ± SEM of the percentage. (F) Comparison between cells transduced with IFITM1–3 and the empty vector was performed using an analysis of the area under the curve of the replicates represented in (E). Values are represented as mean + SEM (n = 6 wells/condition, n = 3 experiments). Statistical analysis was performed with one-way ANOVA to compare treatment to vehicle controls. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Ectopic expression of GFP , FLAG , and IFITMs in EVCT explants transduced with IFITM1–3 was verified by immunofluorescence (G–J) . (G) Representative image of placental explants transduced with GFP or IFITM1–3, cultured on Matrigel, and immunostained for FLAG (yellow), GFP (green), and IFITM1 (red) or IFITM2/3 (magenta), and then counterstained with DAPI (blue). Scale bars: 100 μm. (H) Representative image of EVCT explants transduced with GFP or IFITM1–3, cultured on Matrigel, and immunostained for FLAG (yellow), GFP (green), and ITGA5 (red) or HLAG (magenta), and then counterstained with DAPI (blue). Scale bars: 100 μm. Inhibition of IFITM1-transduced EVCT invasion using villous explant culture model (I–J) . (I) Representative image of transduced extravillous explants cultured on Matrigel and immunostained for integrin α5 (ITGA5, marker of invasion, red). Scale bars: 100 μm. (J) Statistical analysis of the invasion distance of ITGA5 + EVCTs, as representatively shown in I. Values are presented as mean + SD (n = 3 explants per condition, n = 3 placentas). One-way ANOVA was performed to compare treatment to pTRIP-GFP control; ∗∗∗∗p < 0.0001.

Article Snippet: Rabbit anti-Fragilis [IFITM2/3] Monoclonal antibody (clone EPR5242) , Abcam , Cat# ab109429; RRID: AB_10865792.

Techniques: Expressing, Transduction, Western Blot, Immunofluorescence, Flow Cytometry, SDS Page, Plasmid Preparation, Staining, Inhibition, Cell Culture, Marker

Journal: iScience

Article Title: IFITM1 inhibits trophoblast invasion and is induced in placentas associated with IFN-mediated pregnancy diseases

doi: 10.1016/j.isci.2023.107147

Figure Lengend Snippet:

Article Snippet: Rabbit anti-Fragilis [IFITM2/3] Monoclonal antibody (clone EPR5242) , Abcam , Cat# ab109429; RRID: AB_10865792.

Techniques: Recombinant, Electron Microscopy, Selection, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Software