Journal: Stem Cell Research & Therapy

Article Title: Lumican from DESC spheroids enhances odontoblastic differentiation of SCAPs

doi: 10.1186/s13287-025-04849-7

Figure Lengend Snippet: Lumican enhances SCAPs’ odontoblastic differentiation via ITGβ1-ERK pathway ( a , b ) Western blot analysis and quantification of phosphorylation levels of JNK, p38 and ERK in SCAPs following Lumican treatment. Note the time-dependent increase in phosphorylation of both JNK and ERK. Control: 5% DMSO without inhibitor. c Fluorescence microscopy images revealed increased fluorescence intensity following Lumican treatment. d ALP and ARS staining showing that U0126 treatment most significantly abolished the pro-odontoblastic effects of Lumican on SCAPs. Control: 5% DMSO without inhibitor. e RT-qPCR demonstrating that Lumican-induced upregulation of odontogenesis-related markers Dspp , Dmp1 , Ocn and Nestin was most significantly reversed by U0126 treatment. Control: 5% DMSO without inhibitor. f , g Western blot analysis and quantification demonstrating that Lumican-induced upregulation of odontogenesis-related markers DSPP, DMP1 and OCN at both protein levels was reversed by U0126 treatment. Control: 5% DMSO without inhibitor ( n = 3; one-way ANOVA). h , i Western blot analysis and quantification showing increased ITGβ1 expression in Lumican-treated SCAPs. j Fluorescence microscopy images revealing co-localization (yellow) of ITGβ1 (red) and 6*His-tagged Lumican (green) in SCAPs membrane. k Co-IP assay confirming direct interaction between 6*His-tagged Lumican and ITGβ1. l , m Western blot analysis and quantification showing AIIB2 treatment did not affect ITGβ1 expression in SCAPs, but attenuated Lumican-enhanced ERK phosphorylation. n ALP and ARS staining showed that AIIB2 pretreatment counteracted the enhancing effect of Lumican on both early differentiation and late-stage mineralization. o RT-qPCR demonstrating that AIIB2 pretreatment counteracted the Lumican-induced upregulation of odontogenesis-related markers Dspp , Dmp1 , Ocn and Nestin . (p , q) Western blot analysis and quantification demonstrating that AIIB2 pretreatment counteracted the Lumican-induced upregulation of odontogenesis-related markers Dspp , Dmp1 , Ocn and Nestin

Article Snippet: After fixation, permeabilization, and blocking, the cells were incubated with anti-6*His antibody (66005-1-Ig, Proteintech, China, 1:200) and anti-integrin β1 (ITGβ1) antibody (12594-1-AP, Proteintech, China, 1:200) at 4 °C for 16 h. The cells were rinsed with PBS and treated with Alexa Fluor 488-conjugated (AF488, Beyotime, China, 1:200) and Alexa Fluor 647-conjugated secondary antibodies (A0473, Beyotime, China, 1:200) at room temperature for 1 h. The nuclei were counterstained with DAPI for 5 min.

Techniques: Western Blot, Phospho-proteomics, Control, Fluorescence, Microscopy, Staining, Quantitative RT-PCR, Expressing, Membrane, Co-Immunoprecipitation Assay

Journal: BMC Cancer

Article Title: Unveiling the ITGβ1-CLIC1 axis: a new frontier in oral squamous cell carcinoma progression and immune evasion: insights from the tumor-mimetic ECM system

doi: 10.1186/s12885-025-15402-3

Figure Lengend Snippet: Analysis of clinical samples and data from OSCC. A Patients screening process. B Baseline characteristics of patients. C Correlation between ITGβ1 and CLIC1 expression and stage. D Recurrence-free survival analysis

Article Snippet: Specific primary antibodies were used to detect the target proteins: ITGβ1 (1:1000; Wanleibio, Shenyang, China; Rabbit anti-human) and CLIC1 (1:1000; Wanleibio, Shenyang, China; Rabbit anti-human), followed by incubation with secondary antibody (Goat anti-rabbit).

Techniques: Expressing

Journal: Journal of Virology

Article Title: The integrin protein ITGβ1 effectively suppresses porcine epidemic diarrhea virus replication through facilitating MDA5 oligomerization and subsequent activation of the type I interferon signaling pathway

doi: 10.1128/jvi.01553-25

Figure Lengend Snippet: PEDV infection upregulates ITGβ1 expression in both Vero and LLC-PK1 cells. ( A and B ) qPCR was used to measure the expression levels of ITGβ1 mRNA in Vero and LLC-PK1 cells infected with PEDV at different MOIs (0.01, 0.1, 0.5, and 1). ( C and D ) Vero cells were infected with PEDV at an MOI of 0.1 or mock-infection, and ITGβ1 expression was analyzed at 12 and 18 hpi. ( E and F ) Similarly, qPCR and Western blotting analysis were performed to assess the ITGβ1 expression in LLC-PK1 cells. Data are presented as means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-tailed Student’s t -test (for two-group comparisons) or one-way ANOVA (for multi-group comparisons).

Article Snippet: Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) antibody (AC033) and anti-ITGβ1 antibody (A2217 and A21234) were obtained from Abclonal.

Techniques: Infection, Expressing, Western Blot, Two Tailed Test

Journal: Journal of Virology

Article Title: The integrin protein ITGβ1 effectively suppresses porcine epidemic diarrhea virus replication through facilitating MDA5 oligomerization and subsequent activation of the type I interferon signaling pathway

doi: 10.1128/jvi.01553-25

Figure Lengend Snippet: PEDV infection upregulates the expression of ITGβ1 through the mediation of the transcription factor c-Myc. ( A ) HEK293T cells were co-transfected with a series of truncated ITGβ1 promoter plasmids (−2,000 to −1) and the pRL-TK-Luc reporter plasmid, and dual-luciferase activity was analyzed 24 h post-transfection. In the figure, gray represents gene fragments with detectable luciferase activity in the luciferase activity assay, while black indicates gene fragments for which no luciferase activity was detected. ( B ) Regulatory factors within the ITGβ1 promoter region were predicted using the JASPAR vertebrate database ( https://jaspar.genereg.net/ ). ( C ) LLC-PK1 cells were infected with PEDV at an MOI of 0.1 or mock-infected, and the mRNA expression levels of the predicted regulatory factors were detected using qPCR. ( D ) HEK293T cells were co-transfected with the ITGβ1 promoter-driven luciferase vector, plasmids expressing TRMT, TFEB, c-Myc, and TFEB, and the pRL-TK-Luc Renilla luciferase reporter vector. Dual-luciferase activity was measured 24 h post-transfection. ( E ) The expression of ITGβ1 mRNA in LLC-PK1 cells transfected with siRNAs targeting the predicted regulatory factors was assessed using qPCR. Data are shown as means ± SD of triplicate samples. ***, P < 0.001; ****, P < 0.0001; ns, not significant by two-tailed Student’s t -test.

Article Snippet: Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) antibody (AC033) and anti-ITGβ1 antibody (A2217 and A21234) were obtained from Abclonal.

Techniques: Infection, Expressing, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Two Tailed Test

Journal: Journal of Virology

Article Title: The integrin protein ITGβ1 effectively suppresses porcine epidemic diarrhea virus replication through facilitating MDA5 oligomerization and subsequent activation of the type I interferon signaling pathway

doi: 10.1128/jvi.01553-25

Figure Lengend Snippet: Overexpression of ITGβ1 effectively inhibits PEDV replication in both Vero and LLC-PK1 cell lines. ( A ) LLC-PK1 cells were transfected with either ITGβ1 or an empty vector (1 µg). At 24 h post-transfection, cells were infected with PEDV at an MOI of 0.1 or mock-infected. Cells were harvested at 12, 16, and 20 hpi, and the expression of the PEDV N protein was analyzed by Western blotting, with GAPDH as a loading control. ( B and C ) The mRNA levels of the PEDV N protein and viral titers in the cell supernatants collected at 12, 16, and 20 hpi were quantified using qPCR and the TCID 50 assay, respectively. ( D ) Vero cells were transfected with 1 µg of ITGβ1 or an empty vector, followed by PEDV infection (MOI = 0.1) at 24 h post-transfection. Cells were collected at 12, 16, and 20 hpi, and the expression level of the PEDV N protein was assessed by Western blotting. ( E and F ) Vero cells were transfected with 1 µg of ITGβ1 or an empty vector, and PEDV N protein expression and viral titers in supernatants harvested at specified time points were evaluated using qPCR and the TCID 50 assay, respectively. ( G ) Western blotting was performed to evaluate the replication level of the PEDV N protein at 24 hpi in ITGβ1-overexpressing LLC-PK1 ITGβ1 and Vero ITGβ1 cells infected with varying doses of PEDV, with wild-type LLC-PK1 WT and Vero WT cells as controls. ( H ) ITGβ1-overexpressing LLC-PK1 ITGβ1 and Vero ITGβ1 cells, along with their wild-type counterparts (LLC-PK1 WT and Vero WT ), were infected with PEDV (MOI = 0.1). Cells were harvested at indicated time points, and PEDV replication was detected by Western blotting. Data are shown as means ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ns, not significant by two-tailed Student’s t -test (for two-group comparisons) or one-way ANOVA (for multi-group comparisons).

Article Snippet: Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) antibody (AC033) and anti-ITGβ1 antibody (A2217 and A21234) were obtained from Abclonal.

Techniques: Over Expression, Transfection, Plasmid Preparation, Infection, Expressing, Western Blot, Control, Two Tailed Test

Journal: Journal of Virology

Article Title: The integrin protein ITGβ1 effectively suppresses porcine epidemic diarrhea virus replication through facilitating MDA5 oligomerization and subsequent activation of the type I interferon signaling pathway

doi: 10.1128/jvi.01553-25

Figure Lengend Snippet: Suppression of endogenous ITGβ1 expression enhances PEDV replication. ( A ) LLC-PK1 cells were transfected with either a control siRNA (NC) or an ITGβ1-specific siRNA. At 36 h post-transfection, cells were infected with PEDV at an MOI of 0.1. Cell samples were harvested at 12, 16, and 20 hpi, and the expression of PEDV N protein was analyzed by Western blotting following protein extraction. ( B and C ) After 36 h of siRNA transfection, LLC-PK1 cells were infected with PEDV (MOI = 0.1). Supernatants were then collected at 12, 16, and 20 hpi, and the replication levels and viral titers of PEDV in ITGβ1-knockdown LLC-PK1 cells were determined by qPCR and TCID 50 assay, respectively. ( D ) Similarly, the expression of PEDV N protein at 12, 16, and 20 hpi was evaluated by Western blotting in Vero cells with suppressed endogenous ITGβ1 expression. ( E and F ) The replication levels and virus titers of PEDV in ITGβ1-knockeddown Vero cells were measured by qPCR and TCID 50 assay, respectively, at 12, 16, and 20 hpi. ( G and H ) LLC-PK1 WT and LLC-PK1 ΔITGβ1 cells were infected with PEDV (MOI = 0.1), and cells and supernatants were collected at 12, 16, and 20 hpi. The levels of PEDV N protein and mRNA at the indicated time points were then assessed by Western blotting and qPCR, respectively. ( I ) Following infection with PEDV (MOI = 0.001), cell supernatants from LLC-PK1 WT and LLC-PK1 ΔITGβ1 cells were collected at the specified time points, and PEDV titers were determined by TCID 50 assay. Data are shown as means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed Student’s t -test (for two-group comparisons) or one-way ANOVA (for multi-group comparisons).

Article Snippet: Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) antibody (AC033) and anti-ITGβ1 antibody (A2217 and A21234) were obtained from Abclonal.

Techniques: Expressing, Transfection, Control, Infection, Western Blot, Protein Extraction, Knockdown, Virus, Two Tailed Test

Journal: Journal of Virology

Article Title: The integrin protein ITGβ1 effectively suppresses porcine epidemic diarrhea virus replication through facilitating MDA5 oligomerization and subsequent activation of the type I interferon signaling pathway

doi: 10.1128/jvi.01553-25

Figure Lengend Snippet: ITGβ1 is essential for the PEDV-induced antiviral response. ( A ) LLC-PK1 WT and LLC-PK1 ΔITGβ1 cells were infected with PEDV at an MOI of 0.1. Changes in IFN-β mRNA levels were measured by qPCR at 0, 4, 8, and 12 hpi. ( B ) LLC-PK1 WT and LLC-PK1 ΔITGβ1 cells were infected with PEDV (MOI = 0.1), and IFN-β mRNA levels were analyzed by qPCR at 12, 15, 18, and 21 hpi. ( C ) LLC-PK1 WT and LLC-PK1 ΔITGβ1 cells were infected with PEDV at an MOI of 0.1, and cells were collected at 12, 15, 18, and 21 hpi. The cell supernatants were then analyzed by qPCR to determine the IFN-β mRNA levels, as well as the copy number of the PEDV-N protein. ( D ) LLC-PK1 WT and LLC-PK1 ΔITGβ1 cells were inoculated with PEDV (MOI = 0.1) and SeV at 80HAU/mL, respectively. Cells were collected at 18 hpi, and changes in IFN-β mRNA levels were detected by qPCR. ( E ) LLC-PK1 ΔITGβ1 cells were transfected with ITGβ1 or an empty vector, followed by infection with PEDV (MOI = 0.1). At 18 hpi, cells were collected, and IFN-β mRNA levels were analyzed by qPCR. ( F ) LLC-PK1 cells were transfected with negative control (NC) and siRNA, followed by infection with PEDV (MOI = 0.1). Samples were collected at specified time points, and changes in IFN-β mRNA levels were measured by qPCR. Data are shown as means ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant by one-way ANOVA.

Article Snippet: Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) antibody (AC033) and anti-ITGβ1 antibody (A2217 and A21234) were obtained from Abclonal.

Techniques: Infection, Transfection, Plasmid Preparation, Negative Control

Journal: Journal of Virology

Article Title: The integrin protein ITGβ1 effectively suppresses porcine epidemic diarrhea virus replication through facilitating MDA5 oligomerization and subsequent activation of the type I interferon signaling pathway

doi: 10.1128/jvi.01553-25

Figure Lengend Snippet: ITGβ1 promotes SeV-mediated IFN-β production. ( A ) HEK293T cells were co-transfected with ITGβ1 or an empty vector, along with the IFN-β-Luc reporter plasmid and the pRL-TK plasmid. At 24 h post-transfection, cells were either mock-treated or treated with SeV for 12 h. Subsequently, the cells were harvested, and the IFN-β promoter activity was assessed using a dual luciferase assay. ( B ) Cells were co-transfected with ITGβ1 or an empty vector, together with either the IRF3-Luc or NF-κB-Luc reporter plasmid and the pRL-TK plasmid. After SeV stimulation, cells were collected at 12 h post-treatment and analyzed using a dual luciferase assay. ( C ) HEK 293T cells or LLC-PK1 cells were transfected with ITGβ1 or an empty vector (1 µg each). At 24 hours post-transfection, cells were treated with SeV or left untreated for 12 hours. Subsequently, the cell lysates were analyzed by immunoblotting using antibodies specific for phosphorylated IRF3 or p65, as well as total IRF3 or p65. ( D ) LLC-PK1 cells were transfected with si-ITGβ1 or NC. At 24 h post-transfection, the cells were either treated with Sendai virus (SeV) or left untreated for 12 h. Cell lysates were then collected and subjected to Western blotting analysis using antibodies against phosphorylated TBK1 (p-TBK1), IRF3 (p-IRF3) or phosphorylated p65 (P-P65), as well as antibodies against total TBK1, IRF3 or total p65. ( E ) HEK293T cells were transfected with ITGβ1 or an empty vector. At 24 h after transfection, cells were either mock-infected or infected with SeV for 12 h, as described previously. Cells were fixed, permeabilized, and incubated with primary antibodies: rabbit anti-IRF3 or anti-p65, and mouse anti-ITGβ1. Cells were then incubated with goat anti-rabbit 594-conjugated secondary antibody (labeled in red) and goat anti-mouse 488-conjugated secondary antibody (labeled in green). Nuclei were stained with DAPI, and images were acquired using a laser scanning confocal microscope. Data are shown as means ± SD of three independent experiments. **, P < 0.01; ****, P < 0.0001; ns, not significant by one-way ANOVA.

Article Snippet: Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) antibody (AC033) and anti-ITGβ1 antibody (A2217 and A21234) were obtained from Abclonal.

Techniques: Transfection, Plasmid Preparation, Activity Assay, Luciferase, Western Blot, Virus, Infection, Incubation, Labeling, Staining, Microscopy

Journal: Journal of Virology

Article Title: The integrin protein ITGβ1 effectively suppresses porcine epidemic diarrhea virus replication through facilitating MDA5 oligomerization and subsequent activation of the type I interferon signaling pathway

doi: 10.1128/jvi.01553-25

Figure Lengend Snippet: ITGβ1 promotes MDA5-mediated IFN-β production. HEK293T cells were co-transfected with plasmids encoding IFN-β-Luc, pRL-TK (as a control for transfection efficiency), and pcDNA3.1-ITGβ1-HA, along with plasmids expressing RIG-I ( A ), MyD88 ( B ), MDA5 ( C ), MAVS ( D ), TBK1 ( E ), and IKKα ( F ) using plasmid amounts of 0.5, 0.25, and 0.05 µg, respectively. Renilla luciferase activity was used as a normalization control. At 24 h post-transfection, cell lysates were collected, and the activities of each molecule were assessed using a dual luciferase reporter assay. In a separate experiment ( G ), HEK293T cells were transfected with plasmids encoding ITGβ1 (0.5, 1, and 1.5 µg), MDA5 (0.25 µg), or an empty vector, along with pRL-TK (0.05 µg). At 24 h post-transfection, cells were either treated with SeV for 12 h or left untreated. Cell lysates were then collected, and luciferase activity was analyzed using a dual-luciferase reporter assay. Data are shown as means ± SD of least three independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant by two-tailed Student’s t -test (for two-group comparisons) or one-way ANOVA (for multi-group comparisons).

Article Snippet: Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) antibody (AC033) and anti-ITGβ1 antibody (A2217 and A21234) were obtained from Abclonal.

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay, Two Tailed Test

Journal: Journal of Virology

Article Title: The integrin protein ITGβ1 effectively suppresses porcine epidemic diarrhea virus replication through facilitating MDA5 oligomerization and subsequent activation of the type I interferon signaling pathway

doi: 10.1128/jvi.01553-25

Figure Lengend Snippet: ITGβ1 enhances dsRNA-induced IFN-β production. ( A ) HEK293T cells were co-transfected with ITGβ1, MDA5 (or an empty vector serving as a control), the IFN-β-Luc reporter plasmid, and the pRL-TK plasmid using plasmid amounts of 0.5, 0.5, 0.25, and 0.05 µg, respectively. At 24 h post-transfection, cells were either left untreated or stimulated with poly(I:C). Cell lysates were collected at 0, 3, 6, 9, and 12 h post-stimulation, and IFN-β levels were quantified using a dual-luciferase reporter assay. ( B ) Wild-type LLC-PK1 WT cells and ITGβ1-knockout LLC-PK1 ΔITGβ1 cells stimulated with poly(I:C). Cells were harvested at 0, 3, 6, 9, and 12 h post-stimulation, and changes in IFN-β mRNA levels were analyzed by qPCR. ( C ) LLC-PK1 ΔITGβ1 cells were co-transfected with ITGβ1 or an empty vector. At 24 h post-transfection, cells were either stimulated with poly(I:C) or left unstimulated. Cells were collected at 0, 3, 6, 9, and 12 h post-stimulation, and IFN-β mRNA expression was measured by qPCR. ( D ) LLC-PK1 WT and LLC-PK1 ΔITGβ1 cells were co-transfected with ITGβ1, MDA5 (or an empty vector), the IFN-β-Luc reporter plasmid, and the pRL-TK plasmid at transfection ratios of 0.5, 0.5, 0.25, and 0.05 µg, respectively. At 24 h post-transfection, cells were treated with poly(I:C) for 6 h, and IFN-β activity was assessed using a dual-luciferase reporter assay. Data are presented as means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant by two-tailed Student’s t -test (for two-group comparisons) or one-way ANOVA (for multi-group comparisons).

Article Snippet: Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) antibody (AC033) and anti-ITGβ1 antibody (A2217 and A21234) were obtained from Abclonal.

Techniques: Transfection, Plasmid Preparation, Control, Luciferase, Reporter Assay, Knock-Out, Expressing, Activity Assay, Two Tailed Test

Journal: Journal of Virology

Article Title: The integrin protein ITGβ1 effectively suppresses porcine epidemic diarrhea virus replication through facilitating MDA5 oligomerization and subsequent activation of the type I interferon signaling pathway

doi: 10.1128/jvi.01553-25

Figure Lengend Snippet: ITGβ1 interacts with MDA5 and promotes MDA5 oligomerization. ( A ) HEK293T cells were transfected with an empty vector, HA-tagged ITGβ1 or MDA5, and Flag-tagged ITGβ1 or MDA5, either individually or in combination. At 24 h post-transfection, cells were harvested, and co-immunoprecipitation (Co-IP) analysis was performed using anti-Flag or anti-HA magnetic beads. ( B ) After HEK 293T cells reached confluency (formed a monolayer), the cells were harvested. Magnetic beads were incubated with either anti-MDA5 antibody or anti-ITGβ1 antibody, followed by Co-IP analysis. ( C ) HEK293T cells were transfected with an empty vector, pcDNA3.1-ITGβ1-HA, and pcDNA3.1-MDA5-Flag. After 24 h, the cells were permeabilized, fixed, and stained with rabbit anti-HA (red) and mouse anti-Flag (green) primary antibodies. Cells were then incubated with goat anti-rabbit 594-conjugated (red) and goat anti-mouse 488-conjugated (green) secondary antibodies, followed by DAPI staining. Images were visualized using laser confocal microscopy. ( D ) HEK 293T cells were fixed and permeabilized, then incubated with primary antibodies: mouse anti-MDA5 antibody (green) and rabbit anti-ITGβ1 antibody (red). Subsequent incubation was performed with secondary antibodies: goat anti-rabbit IgG conjugated to Alexa Fluor 594 (red) and goat anti-mouse IgG conjugated to Alexa Fluor 488 (green). After DAPI staining, the cells were observed under a laser confocal microscope. ( E ) The structural domains of ITGβ1 were predicted using the SMART online analysis tool ( http://smart.embl-heidelberg.de/ ). Truncated constructs were generated based on the results, with the dark gray region representing the transmembrane domain, flanked by the extracellular domain and cytoplasmic tail, respectively. ( F ) HEK293T cells were co-transfected with pCDNA3.1-MDA5-Flag and either an empty vector or full-length/truncated ITGβ1 plasmids. At 24 h post-transfection, cells were lysed, and Co-IP was performed using anti-Flag antibody-conjugated magnetic beads, followed by protein immunoblotting analysis. ( G ) HEK293T cells were transfected or co-transfected with an empty vector, HA-tagged ITGβ1, and Flag-tagged MDA5. After 24 h, native PAGE and denaturing PAGE were employed for Western blotting analysis to detect exogenous MDA5 oligomerization using a Flag antibody. ( H ) LLC-PK1 cells were transfected or co-transfected with empty vector, HA-tagged ITGβ1, Flag-tagged MDA5, or si-ITGβ1, respectively. At 24 h post-transfection, the cells were stimulated with poly(I:C) (2 µg/mL). Cell samples were collected at 3, 6, and 12 h post-stimulation, subjected to disuccinimidyl suberate (DSS) cross-linking, and then analyzed for MDA5 oligomerization by SDS-PAGE and Western blotting. ( I ) HEK 293T cells were transfected or co-transfected with empty vector, HA-tagged ITGβ1, or Flag-tagged MDA5, respectively. At 24 h post-transfection, the cells were stimulated with poly(I:C) (2 µg/mL) for 12 h. After stimulation, cell samples were collected, treated with DSS cross-linking, and MDA5 oligomerization was detected by SDS-PAGE and Western blotting. ( J ) HEK293T cells were transfected with an empty vector, ITGβ1, MDA5, IFN-β-Luc plasmid, and pRL-TK plasmid at ratios of 0.5, 0.5, 0.25, and 0.05 µg, respectively. At 24 h post-transfection, cells were stimulated with poly(I:C) or left unstimulated. After 12 h, cells were collected, and IFN-β activation was measured using a dual luciferase reporter system. ( K ) A schematic diagram illustrating the truncation of MDA5 is provided. ( L ) HEK293T cells were co-transfected with an empty vector, HA-tagged ITGβ1, and Flag-tagged full-length MDA5 or various truncated MDA5 fragments. At 24 h post-transfection, Co-IP was performed using anti-HA magnetic beads, followed by Western blotting analysis. ( M ) HEK293T cells were co-transfected with pcDNA3.1, pcDNA3.1-ITGβ1-HA, and pcDNA3.1-MDA5 (2CARD)-Flag at ratios of 0.5, 0.5, and 0.5 µg, respectively. At 24 h post-transfection, cell lysates were collected, and MDA5 oligomerization was detected by native PAGE and denaturing PAGE using immunoblotting techniques. Data are presented as means ± SD from three independent experiments. **, P < 0.01; ***, P < 0.001 by one-way ANOVA.

Article Snippet: Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) antibody (AC033) and anti-ITGβ1 antibody (A2217 and A21234) were obtained from Abclonal.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Co-Immunoprecipitation Assay, Magnetic Beads, Incubation, Staining, Confocal Microscopy, Microscopy, Construct, Generated, Western Blot, Clear Native PAGE, SDS Page, Activation Assay, Luciferase

Journal: Journal of Virology

Article Title: The integrin protein ITGβ1 effectively suppresses porcine epidemic diarrhea virus replication through facilitating MDA5 oligomerization and subsequent activation of the type I interferon signaling pathway

doi: 10.1128/jvi.01553-25

Figure Lengend Snippet: Mechanism of ITGβ1-mediated anti-PEDV activity. Following the infection of host cells by PEDV, the host cells activate the transcription factor c-Myc, which upregulates the expression of ITGβ1. The intracellular domain of ITGβ1 can interact with the 2CARD region of MDA5, promoting its oligomerization and subsequent activation. Activation of MDA5 initiates the phosphorylation and nuclear translocation of IRF3 and NF-κB, leading to the induction of IFN-β production.

Article Snippet: Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) antibody (AC033) and anti-ITGβ1 antibody (A2217 and A21234) were obtained from Abclonal.

Techniques: Activity Assay, Infection, Expressing, Activation Assay, Phospho-proteomics, Translocation Assay