Fig 1: LECT2 is a positive regulator of RIG-I-mediated innate immune responses.a qRT-PCR analysis of LECT2 mRNA in HepG2-WT and HepG2-LECT2 cells. Results were normalised to those of ACTB. b qRT-PCR analysis of IFNB1 mRNA in HepG2-WT and HepG2-LECT2 cells at 12 h after mock (Cont) or HCV-RNA transfection. Data shown are means of 3 technical replicates ± SEM. p = 0.001 by two-way ANOVA with Tukey’s multiple comparison test. c Immunoblot analysis of RIG-I, MDA5, p-IRF3, IRF3, LECT2 and β-actin in HepG2-WT and HepG2-LECT2 cells at 6 h after mock (Cont) or HCV-RNA transfection. d qRT-PCR analysis of IFNB1, MX1 and OAS2 in non-treated (Nont) and r-LECT2-treated HepG2 cells at 0, 3, 6 and 12 h after HCV-RNA transfection. Data shown are means of 2 technical replicates. e qRT-PCR analysis of IFNB1, MX1 and OAS2 in non-treated (Nont), proteinase-treated r-LECT2 (r-LECT2 + proteinase), and r-LECT2-treated cells (Cont siRNA-transfected HepG2 cells, RIG-I siRNA-transfected HepG2 cells, and MDA5 siRNA-transfected HepG2 cells) at 12 h after HCV-RNA transfection. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001, not significant (ns) by two-way ANOVA with Tukey’s multiple comparison test. f Immunoblot analysis of RIG-I, MDA5, and β-actin in Nont, r-LECT2 + proteinase and r-LECT2-treated cells at 12 h after HCV-RNA transfection. g 50 ng/mL r-LECT2 and LECT2 specific antibody (0, 50 and 100 ng/mL) were reacted for 15 min at room temperature, and then their reactants were treated with HepG2 cells for 1 h and then transfected with HCV-RNA. (upper) qRT-PCR analysis of IFNB1 at 12 h after HCV-RNA transfection. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001 by two-way ANOVA with Tukey’s multiple comparison test. (lower) Immunoblot analysis of RIG-I, MDA5, p-IRF3, IRF3, LECT2 and β-actin at 12 h after HCV-RNA transfection. Source data are provided as a Source data file.
Fig 2: PTP4A1 recruitment to MET is essential for LECT2-mediated RIG-I dependent innate immune responses.a HepG2 cells were treated with r-LECT2 (0, 5, 10, 50 ng/mL). At 6 h after treatment, lysates were immunoprecipitated with an anti-MET antibody. Immunoblot analysis of MET, p-MET (Tyr1349), p-SHP2, SHP2, PTP4A1, c-Cbl and β-actin in immunoprecipitated samples and input cell lysate. b HepG2-MET-KO cells were transfected with Control plasmid (Cont), MET WT plasmid, MET Y1349F plasmid, or PTP4A1 plasmid. At 48 h after transfection, lysates were immunoprecipitated with an anti-MET antibody. Immunoblot analysis of MET, p-MET (Tyr1349), p-SHP2, SHP2, PTP4A1, c-Cbl and β-actin in immunoprecipitated samples and input cell lysate. c HepG2 cells were transfected with 50 nM c-Cbl siRNA. The concentration of siRNA was adjusted to 50 nM with control siRNA. At 24 h after siRNA transfection, siRNA-transfected HepG2 cells were treated with 50 ng/mL r-LECT2 for 1 h and then transfected with HCV-RNA. c (upper) Immunoblot analysis of c-Cbl, RIG-I and β-actin at 12 h after HCV-RNA transfection. c (lower) qRT-PCR analysis of IFNB1 at 12 h after HCV-RNA transfection. Results were normalised to those of ACTB. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001, not significant (ns) by two-way ANOVA with Tukey’s multiple comparison test. d HepG2-MET-KO cells were transfected with Control plasmid or MET plasmid; at 48 h after transfection, the cells were treated with 50 ng/mL r-LECT2 and/or 10 ng/mL HGF. At 1 h after r-LECT2 and/or HGF treatment, lysates were immunoprecipitated with an anti-MET antibody. Immunoblot analysis of MET, p-MET (Tyr1234/1235), p-MET (Tyr1349), p-MET (Tyr1356), PTP4A1, p-SHP2, SHP2 and β-actin in immunoprecipitated samples and input cell lysate. Source data are provided as a Source data file.
Fig 3: MET is essential for LECT2-mediated innate immune responses.a HepG2 cells were transfected with FLAG-tagged LECT2 plasmid and MET plasmid. Co-immunofluorescence staining of LECT2 using an anti-FLAG antibody and MET using an anti-MET antibody in HepG2 cells. DAPI was used to stain nuclei. b qRT-PCR analysis of IFNB1 in MET-knockdown HepG2 cells at 12 h after HCV-RNA transfection in the presence or absence of r-LECT2. Results were normalised to those of ACTB. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001, **p = 0.002, not significant (ns) by two-way ANOVA with Tukey’s multiple comparison test. c Immunoblot analysis of MET, RIG-I, p-IRF3 and β-actin in MET-knockdown HepG2 cells at 12 h after HCV-RNA transfection in the presence or absence (Nont) of r-LECT2. d Immunoblot analysis of MET and GAPDH in HepG2-WT and HepG2-MET-KO cells. e (left) qRT-PCR analysis of IFNB1 in HepG2-WT and HepG2-MET-KO cells at 12 h after miR-122 and H77S.3/GLuc-RNA co-transfection in the presence or absence (Nont) of r-LECT2. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001, not significant (ns) by two-way ANOVA with Tukey’s multiple comparison test. e (right) GLuc activity in HepG2-WT and HepG2-MET-KO cells at 12 h after miR-control (miR-cont) or miR-122 and H77S.3/GLuc-RNA co-transfection in the presence or absence (Nont) of r-LECT2. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001, not significant (ns) by two-way ANOVA with Tukey’s multiple comparison test. f qRT-PCR analysis of IFNB1 and EMCV in HepG2-WT and HepG2-MET-KO cells at 24 h after EMCV infection in the presence or absence (Nont) of r-LECT2. Data shown are means of 3 technical replicates ± SEM. Not significant (ns) by two-way ANOVA with Tukey’s multiple comparison test. Source data are provided as a Source data file.
Fig 4: SHP2 inactivation by LECT2 is essential for LECT2-mediated RIG-I dependent innate immune activation.a HepG2 cells were treated with r-LECT2. Immunoblot analysis of p-SHP2, SHP2, p-GAB1, GAB1 and β-actin in r-LECT2-treated HepG2 cells. b Relative intensity. c Immunoblot analysis of K48-ubiquitin (Ub), RIG-I (FLAG), LECT2 (Myc) and β-actin in immunoprecipitated samples and input cell lysate. d qRT-PCR analysis of IFNB1 in HepG2-WT and HepG2-SHP2-KO cells at 12 h after Cont or Poly(I:C) transfection in the presence or absence of r-LECT2. Results were normalised to those of ACTB. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001, not significant (ns) by two-way ANOVA with Tukey’s multiple comparison test. e Immunoblot analysis of SHP2, RIG-I, MDA5, p-IRF3, IRF3 and β-actin in HepG2-WT and HepG2-SHP2-KO cells at 12 h after Cont or Poly(I:C) transfection in the presence or absence of r-LECT2. f Immunoblot analysis of RIG-I, MDA5, p-IRF3, IRF3, p-ERK, ERK1/2 and β-actin in DMSO-treated HepG2 cells and SHP099-treated HepG2 cells at 12 h after HCV-RNA transfection in the presence or absence of r-LECT2. g qRT-PCR analysis of IFNB1, DDX58, IFIH1, OAS2 and MX1 in DMSO-treated HepG2 cells and SHP099-treated HepG2 cells at 12 h after HCV-RNA transfection in the presence or absence of r-LECT2. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001, not significant (ns) by two-way ANOVA with Tukey’s multiple comparison test. h Immunoblot analysis of K48-Ub, SHP2, LECT2 (FLAG), RIG-I and β-actin in immunoprecipitated samples and input cell lysate. i HepG2-RIG-I-KO cells were transfected with RIG-I plasmid, SHP2 plasmid and FLAG-tagged LECT2 plasmid. At 48 h after transfection, the cells were transfected with HCV-RNA. qRT-PCR analysis of IFNB1 in these cells at 12 h after HCV-RNA transfection. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001, **p = 0.001 by two-way ANOVA with Tukey’s multiple comparison test. Source data are provided as a Source data file.
Fig 5: PTP4A1 is essential for the enhancement of innate immune response by LECT2.a Immunoblot analysis of MET, PTP4A1, p-SHP2, RIG-I and β-actin in immunoprecipitated samples and input cell lysate. b HepG2-MET-KO cells were transfected with Control, MET and PTP4A1 plasmid or co-transfected with MET and PTP4A1 plasmid. These cells were treated with 50 ng/mL r-LECT2 for 1 h and then transfected with HCV-RNA. qRT-PCR analysis of IFNB1 in these cells. Results were normalised to those of ACTB. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001, not significant (ns) by two-way ANOVA with Tukey’s multiple comparison test. c–e KH-MET-KO cells were transfected with Control, MET and PTP4A1 plasmid or co-transfected with MET and PTP4A1 plasmid. These cells were treated with 50 ng/mL r-LECT2 for 1 h and then transfected with H77S.3/GLuc-RNA. c Immunoblot analysis of MET, PTP4A1 and β-actin in these cells. d qRT-PCR analysis of IFNB1, DDX58 and IFIH1 in these cells. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001, **p = 0.002, not significant (ns) by two-way ANOVA with Tukey’s multiple comparison test. e GLuc activity in these cells at 48 h after H77S.3/GLuc-RNA transfection. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001, **p = 0.002, not significant (ns) by two-way ANOVA with Tukey’s multiple comparison test. f (left) Co-immunofluorescence staining of MET and PTP4A1 in HepG2 cells. DAPI was used to stain nuclei. f (right) Co-localization was estimated using Pearson’s correlation coefficient and Manders’ co-localization coefficients M1 (red per green) and M2 (green per red). Boxplots: centre line, median; box limits, 25 to 75th percentiles; whiskers, min to max. g (left) Immunoblot analysis of PTP4A1, RIG-I and β-actin at 12 h after HCV-RNA transfection. g (right) qRT-PCR analysis of IFNB1 in HepG2-WT and HepG2-PTP4A1-KO cells at 12 h after HCV-RNA transfection. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001, not significant (ns) by two-way ANOVA with Tukey’s multiple comparison test. Source data are provided as a Source data file.
Supplier Page from Abcam for Anti-LECT2 antibody [OTI2A11]