Journal: The Journal of Clinical Investigation

Article Title: Endoplasmic reticulum–associated degradation is required for nephrin maturation and kidney glomerular filtration function

doi: 10.1172/JCI143988

Figure Lengend Snippet: (A–E) Representative confocal images of nephrin-KDEL (A), nephrin-ZO1 (B), podocin-KDEL (C), podocin-ZO1 (D), and CD2AP-ZO1 (E) in kidney sections from 3-week-old mice (n = 3 mice each). Arrows mark the perinuclear localization of nephrin (A), colocalization of nephrin with ZO1 (B), localization of podocin at the slit diaphragm (C), and colocalization of podocin and ZO1 (D) and CD2AP and ZO1 (E). Images with artificially enhanced KDEL signal are shown in Supplemental Figure 6A. (F) Western blot analysis of nephrin in kidney lysates from 3-, 5-, and 7-week-old mice and (G) quantitation of the percentage of b form nephrin in total nephrin. n = 10 mice each at 3 weeks; n = 10 Sel1Lfl/fl and n = 12 Sel1LPodCre at 5 weeks; and n = 4 each at 7 weeks. Seven-week-old Ire1aPodCre mice were included as a control (n = 3), and the original data are shown in Supplemental Figure 7A. Values represent the mean ± SEM. ***P < 0.001, by 2-tailed Student’s t test (3- and 5-week-old mice) and 1-way ANOVA (7-week-old mice). (H) Western blot analysis of nephrin in EndoH-treated kidney lysates from 5-week-old mice, with quantitation shown in Supplemental Figure 6D (n = 5 mice/group). r, EndoH-resistant form; s, EndoH-sensitive form.

Article Snippet: The following antibodies were used: SEL1L (Abcam, ab78298; 1:1000 for Western blotting); podocin (MilliporeSigma, P0372; 1:100 for immunostaining and ABclonal, A17337; 1:3000 for Western blotting); HRD1 (Proteintech, 13473-1-AP; 1:3000 for Western blotting); synaptopodin (Santa Cruz Biotechnology, sc-515842; 1:100 for immunostaining and ABclonal, A12049; 1:2000 for Western blotting); nephrin (ABclonal, A3048; 1:2000 for Western blotting, 1:100 for immunostaining); KDEL (Abcam, ab12223; 1:200 for immunostaining); ZO1 (Thermo Fisher Scientific, 33-9100; 1:100 for immunostaining); BiP (Abcam, ab21685; 1:5000 for Western blotting, 1:200 for immunostaining); CD2AP (Proteintech, 51046-1-AP; 1:300 for immunostaining); ubiquitin (Ub) (Santa Cruz Biotechnology, sc-8017; 1:1000 for Western blotting); Myc (MilliporeSigma, C3956; 1:5000 for Western blotting); Flag (MilliporeSigma, F-1804; 1:5000 for Western blotting); HSP90 (Santa Cruz Biotechnology, sc-13119; 1:5000 for Western blotting); and histone H2A (Cell Signaling Technology, 2578; 1:2000 for Western blotting).

Techniques: Western Blot, Quantitation Assay

Journal: The Journal of Clinical Investigation

Article Title: Endoplasmic reticulum–associated degradation is required for nephrin maturation and kidney glomerular filtration function

doi: 10.1172/JCI143988

Figure Lengend Snippet: (A) Western blot analysis following nephrin immunoprecipitation in kidney tissues from 5-week-old mice, showing the interaction between nephrin and BiP in the absence of ERAD. (B) Western blot analysis following HRD1 deletion in the HRD1–/– human podocyte line. CON, control. (C) Representative confocal images of nephrin and KDEL staining in human podocytes (n = 5 WT and n = 6 HRD1–/– cells). Scale bars: 5 μm. (D) Western blot analysis of nephrin in transfected WT and HRD1–/– HEK293T cells, digested with or without PNGase F (P) or EndoH (E), with quantitation of the percentage of EndoH-resistant and EndoH-sensitive forms shown below. (E) 35S pulse (30-min) chase (0, 1, 2, and 4 hours) analysis of nascent nephrin protein in HEK293T cells, and (F) quantitation of the percentage of a form nephrin in total nephrin. (G) Western blot analysis of Myc immunoprecipitates in transfected HEK293T cells, treated or not with 10 μM MG132 for 5 hours prior to harvesting, showing ERAD-mediated ubiquitination of nephrin. (H) Western blot analysis of nephrin protein decay in transfected HEK293T cells treated with brefeldin A and/or CHX for the indicated durations, with quantitation from 4 independent experiments shown below. (I) Western blot analysis of nephrin in transfected WT and Hrd1–/– N2a cells under nonreducing or reducing conditions, with the level of HMW nephrin normalized to total nephrin from 3 independent experiments shown below the blot. Data are representative of at least 3 independent experiments. Values represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed Student’s t test.

Article Snippet: The following antibodies were used: SEL1L (Abcam, ab78298; 1:1000 for Western blotting); podocin (MilliporeSigma, P0372; 1:100 for immunostaining and ABclonal, A17337; 1:3000 for Western blotting); HRD1 (Proteintech, 13473-1-AP; 1:3000 for Western blotting); synaptopodin (Santa Cruz Biotechnology, sc-515842; 1:100 for immunostaining and ABclonal, A12049; 1:2000 for Western blotting); nephrin (ABclonal, A3048; 1:2000 for Western blotting, 1:100 for immunostaining); KDEL (Abcam, ab12223; 1:200 for immunostaining); ZO1 (Thermo Fisher Scientific, 33-9100; 1:100 for immunostaining); BiP (Abcam, ab21685; 1:5000 for Western blotting, 1:200 for immunostaining); CD2AP (Proteintech, 51046-1-AP; 1:300 for immunostaining); ubiquitin (Ub) (Santa Cruz Biotechnology, sc-8017; 1:1000 for Western blotting); Myc (MilliporeSigma, C3956; 1:5000 for Western blotting); Flag (MilliporeSigma, F-1804; 1:5000 for Western blotting); HSP90 (Santa Cruz Biotechnology, sc-13119; 1:5000 for Western blotting); and histone H2A (Cell Signaling Technology, 2578; 1:2000 for Western blotting).

Techniques: Western Blot, Immunoprecipitation, Staining, Transfection, Quantitation Assay

Journal: Frontiers in Microbiology

Article Title: Echinococcus granulosus sensu lato promotes osteoclast differentiation through DUSP4-MAPK signaling in osseous echinococcosis

doi: 10.3389/fmicb.2025.1558603

Figure Lengend Snippet: PSC intervention downregulates DUSP4 and upregulates the MAPK signaling pathway. (A) mRNA expression levels of DUSP2 and DUSP4 following different doses of PSC intervention. (B) Protein expression levels of DUSP4 after different doses of PSC intervention. (C) Quantification of DUSP4 protein levels shown in (B) . (D) Visualization of F-actin ring formation and DUSP4 expression in osteoclasts after different doses of PSC intervention. Green: F-actin; red: DUSP4; blue: DAPI. Magnification ×40. (E) Quantification of the F-actin ring area and the absolute and relative fluorescence intensity of DUSP4 shown in (D) . Data represent three independent experiments. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) blocking solution (BIOTEK, China) for 2 h, and then incubated overnight at 4°C with primary antibodies: DUSP4 (1:1000; Boster, #OT17C11); GAPDH (1:2000; Boster, #BG-7) ERK (1:2,000; #4695), p-ERK (1:1,000; #4370), JNK (1:2,000; #9252), p-JNK (1:1,000; #4668), p38 (1:2,000; #8690), p-p38 (1:1,000; #4511) (Cell Signaling Technology, Inc.); MMP-9 (1:1000; #ab76003); c-Fos (1:2,000; #ab190289), TRAP (1:1,000; #ab191406), NFATc1 (4 μg/mL; #ab2796), Cathepsin K (1:2,000; #ab19027) (Abcam, Inc).

Techniques: Expressing, Fluorescence

Journal: Frontiers in Microbiology

Article Title: Echinococcus granulosus sensu lato promotes osteoclast differentiation through DUSP4-MAPK signaling in osseous echinococcosis

doi: 10.3389/fmicb.2025.1558603

Figure Lengend Snippet: Overexpression of DUSP4 affects osteoclast formation. (A) mRNA expression levels of DUSP4 in control, lentiviral-transfected null, and DUSP4-overexpression groups. (B) Protein levels of DUSP4 in the same groups. (C) Quantification of (B) . (D) Cell viability assay in the same groups. (E) TRAP staining of osteoclasts in control and DUSP4-overexpression groups after PSC intervention. Magnification ×40. (F) Quantification of the area occupied by TRAP-positive cells and the number of TRAP-positive cells shown in (E) . (G) F-actin ring staining in control and DUSP4-overexpression groups after PSC intervention green: F-actin; blue: DAPI. Magnification ×40. (H) Quantification of the F-actin ring area and fluorescence intensity shown in (G) . Data represent three independent experiments, and significance was determined using one-way ANOVA. ns, no significance; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) blocking solution (BIOTEK, China) for 2 h, and then incubated overnight at 4°C with primary antibodies: DUSP4 (1:1000; Boster, #OT17C11); GAPDH (1:2000; Boster, #BG-7) ERK (1:2,000; #4695), p-ERK (1:1,000; #4370), JNK (1:2,000; #9252), p-JNK (1:1,000; #4668), p38 (1:2,000; #8690), p-p38 (1:1,000; #4511) (Cell Signaling Technology, Inc.); MMP-9 (1:1000; #ab76003); c-Fos (1:2,000; #ab190289), TRAP (1:1,000; #ab191406), NFATc1 (4 μg/mL; #ab2796), Cathepsin K (1:2,000; #ab19027) (Abcam, Inc).

Techniques: Over Expression, Expressing, Control, Transfection, Viability Assay, Staining, Fluorescence

Journal: Frontiers in Microbiology

Article Title: Echinococcus granulosus sensu lato promotes osteoclast differentiation through DUSP4-MAPK signaling in osseous echinococcosis

doi: 10.3389/fmicb.2025.1558603

Figure Lengend Snippet: Effects of DUSP4 on the MAPK pathway and osteoclast-related genes. (A) Western blot analysis showing changes in MAPK signaling pathway components (p-JNK, p-ERK, and p-p38) in osteoclasts after PSC intervention in control and DUSP4-overexpression groups. (B) Quantification of MAPK pathway phosphorylation levels in osteoclasts from control and DUSP4-overexpression groups, as shown in (A) . (C) mRNA expression levels of osteoclast-related proteins (DUSP4, MMP9, NFATc1, TRAP, and CTSK) after PSC intervention in control and DUSP4-overexpression groups. (D) Western blot analysis of osteoclast-related proteins (DUSP4, MMP9, NFATc1, CTSK, and c-fos) after PSC intervention in control and overexpression groups. (E) Quantification of osteoclast-related proteins expression levels in (D) . Data represent three independent experiments, and significance was determined using one-way ANOVA. ns, no significance; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) blocking solution (BIOTEK, China) for 2 h, and then incubated overnight at 4°C with primary antibodies: DUSP4 (1:1000; Boster, #OT17C11); GAPDH (1:2000; Boster, #BG-7) ERK (1:2,000; #4695), p-ERK (1:1,000; #4370), JNK (1:2,000; #9252), p-JNK (1:1,000; #4668), p38 (1:2,000; #8690), p-p38 (1:1,000; #4511) (Cell Signaling Technology, Inc.); MMP-9 (1:1000; #ab76003); c-Fos (1:2,000; #ab190289), TRAP (1:1,000; #ab191406), NFATc1 (4 μg/mL; #ab2796), Cathepsin K (1:2,000; #ab19027) (Abcam, Inc).

Techniques: Western Blot, Control, Over Expression, Phospho-proteomics, Expressing

Journal: Frontiers in Microbiology

Article Title: Echinococcus granulosus sensu lato promotes osteoclast differentiation through DUSP4-MAPK signaling in osseous echinococcosis

doi: 10.3389/fmicb.2025.1558603

Figure Lengend Snippet: DUSP4 inhibits osteoclasts and attenuates bone destruction in osseous echinococcosis. (A) X-ray examination of long bones from mice treated with empty vector, lentiviral DUSP4, or left untreated in the affection of PSC after 6 months, with red rectangle indicating cyst. (B) Micro-CT images of long bones from each treatment group, with red arrows indicating bone defects. (C) HE staining of bone tissue sections from each treatment group. Magnification ×20. (D) Western blot analysis of osteoclast-related proteins (DUSP4 and CTSK) after PSC intervention in control and DUSP4-overexpression groups. (E) Quantification of osteoclast-related protein expression levels in osteoclasts from control and DUSP4-overexpression groups, as shown in (D) . Data are presented as mean ± SD from five mice per group. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) blocking solution (BIOTEK, China) for 2 h, and then incubated overnight at 4°C with primary antibodies: DUSP4 (1:1000; Boster, #OT17C11); GAPDH (1:2000; Boster, #BG-7) ERK (1:2,000; #4695), p-ERK (1:1,000; #4370), JNK (1:2,000; #9252), p-JNK (1:1,000; #4668), p38 (1:2,000; #8690), p-p38 (1:1,000; #4511) (Cell Signaling Technology, Inc.); MMP-9 (1:1000; #ab76003); c-Fos (1:2,000; #ab190289), TRAP (1:1,000; #ab191406), NFATc1 (4 μg/mL; #ab2796), Cathepsin K (1:2,000; #ab19027) (Abcam, Inc).

Techniques: Plasmid Preparation, Micro-CT, Staining, Western Blot, Control, Over Expression, Expressing

Journal: Frontiers in Microbiology

Article Title: Echinococcus granulosus sensu lato promotes osteoclast differentiation through DUSP4-MAPK signaling in osseous echinococcosis

doi: 10.3389/fmicb.2025.1558603

Figure Lengend Snippet:

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) blocking solution (BIOTEK, China) for 2 h, and then incubated overnight at 4°C with primary antibodies: DUSP4 (1:1000; Boster, #OT17C11); GAPDH (1:2000; Boster, #BG-7) ERK (1:2,000; #4695), p-ERK (1:1,000; #4370), JNK (1:2,000; #9252), p-JNK (1:1,000; #4668), p38 (1:2,000; #8690), p-p38 (1:1,000; #4511) (Cell Signaling Technology, Inc.); MMP-9 (1:1000; #ab76003); c-Fos (1:2,000; #ab190289), TRAP (1:1,000; #ab191406), NFATc1 (4 μg/mL; #ab2796), Cathepsin K (1:2,000; #ab19027) (Abcam, Inc).

Techniques: