Journal: International Journal of Medical Sciences

Article Title: Upregulation of C/EBP Homologous Protein induced by ER Stress Mediates Epithelial to Myofibroblast Transformation in ADTKD-UMOD

doi: 10.7150/ijms.65036

Figure Lengend Snippet: UMOD mutations in patients

Article Snippet: The primary antibodies used were: polyclonal sheep anti-human UMOD antibody (R&D Systems, USA; Cat #AF6144, 1:100), proximal tubule brush border labeling antibody LTL-488 (Fluorescein Lotus Lectin, Vector Laboratories, USA, FL-1321; 1:1000), anti-GRP78 BiP antibody (abcam, Cambridge, UK; ab21685; 1:500) and CHOP (L63F7) mouse mAb (Cell Signaling Technology, Danvers, MA, USA; #2895; 1:50).

Techniques: Mutagenesis, Variant Assay, Sequencing

Journal: International Journal of Medical Sciences

Article Title: Upregulation of C/EBP Homologous Protein induced by ER Stress Mediates Epithelial to Myofibroblast Transformation in ADTKD-UMOD

doi: 10.7150/ijms.65036

Figure Lengend Snippet: Clinical features of patients with UMOD mutations

Article Snippet: The primary antibodies used were: polyclonal sheep anti-human UMOD antibody (R&D Systems, USA; Cat #AF6144, 1:100), proximal tubule brush border labeling antibody LTL-488 (Fluorescein Lotus Lectin, Vector Laboratories, USA, FL-1321; 1:1000), anti-GRP78 BiP antibody (abcam, Cambridge, UK; ab21685; 1:500) and CHOP (L63F7) mouse mAb (Cell Signaling Technology, Danvers, MA, USA; #2895; 1:50).

Techniques: Biomarker Discovery, Immunofluorescence, Microscopy

Journal: International Journal of Medical Sciences

Article Title: Upregulation of C/EBP Homologous Protein induced by ER Stress Mediates Epithelial to Myofibroblast Transformation in ADTKD-UMOD

doi: 10.7150/ijms.65036

Figure Lengend Snippet: Representative histopathology findings of an ADTKD-UMOD renal biopsy (from CASE 2). (A, B) Hematoxylin-Eosin staining showing eosinophilic “fluffy” inclusions in thick ascending limb of Henle's loop (TALH) (arrow), i.e., abnormal protein accumulation. (C, D) Masson's trichrome staining showing interstitial fibrosis changes, obvious fibrosis around the distal tubules and an intracellular hyaline change (arrow). (E-G) Electron microscopy images; (E) a complete image of a distal tubular epithelium. (F, G) partial enlargement of (E) , showing that the rough ER and the smooth ER are obviously expanded, and accumulation of lower electron density in the ER, i.e., abnormal accumulation of protein.

Article Snippet: The primary antibodies used were: polyclonal sheep anti-human UMOD antibody (R&D Systems, USA; Cat #AF6144, 1:100), proximal tubule brush border labeling antibody LTL-488 (Fluorescein Lotus Lectin, Vector Laboratories, USA, FL-1321; 1:1000), anti-GRP78 BiP antibody (abcam, Cambridge, UK; ab21685; 1:500) and CHOP (L63F7) mouse mAb (Cell Signaling Technology, Danvers, MA, USA; #2895; 1:50).

Techniques: Histopathology, Staining, Electron Microscopy

Journal: International Journal of Medical Sciences

Article Title: Upregulation of C/EBP Homologous Protein induced by ER Stress Mediates Epithelial to Myofibroblast Transformation in ADTKD-UMOD

doi: 10.7150/ijms.65036

Figure Lengend Snippet: Uromodulin expression increases in kidneys of ADTKD-UMOD cases. Representative staining images of kidney paraffin sections from a healthy donor (HNK), sporadic chronic interstitial nephritis (CIN) case and two ADTKD-UMOD cases (CASE1, CASE2). Red, uromodulin; green, lotus tetragonolobus lectin (LTL; a marker of the brush border). Original magnification: 400×, scale bar, 20 µm; the areas in the white boxes were enlarged 4×. In HNK, uromodulin was mainly located on the cell membrane of distal tubular epithelial cells. In CIN, uromodulin was also distributed on the cell membrane, and the expression level did not significantly change compared with HNK. Secreted uromodulin protein was seen in the lumen in HNK and CIN (shown by arrows). In ADTKD-UMOD CASE1 and CASE2, there was significant uromodulin expression that aggregated in the cytoplasm of the distal tubular epithelial cells. Secreted uromodulin was not detected in ADTKD-UMOD.

Article Snippet: The primary antibodies used were: polyclonal sheep anti-human UMOD antibody (R&D Systems, USA; Cat #AF6144, 1:100), proximal tubule brush border labeling antibody LTL-488 (Fluorescein Lotus Lectin, Vector Laboratories, USA, FL-1321; 1:1000), anti-GRP78 BiP antibody (abcam, Cambridge, UK; ab21685; 1:500) and CHOP (L63F7) mouse mAb (Cell Signaling Technology, Danvers, MA, USA; #2895; 1:50).

Techniques: Expressing, Staining, Marker, Membrane

Journal: International Journal of Medical Sciences

Article Title: Upregulation of C/EBP Homologous Protein induced by ER Stress Mediates Epithelial to Myofibroblast Transformation in ADTKD-UMOD

doi: 10.7150/ijms.65036

Figure Lengend Snippet: Schematic diagram of CHOP promoting renal interstitial fibrosis in ADTKD-UMOD. Under normal physiological conditions, UMOD is synthesized and transported to the Golgi apparatus for processing, and then transported to the cell membrane. However, in ADTKD-UMOD, when mutant UMOD accumulates in the ER, it causes ER stress, which activates the ER membrane sensors. Consequently, GRP78 separates from the sensors and binds to the mutant UMOD to promote its folding; simultaneously the stress signal is transferred to the nucleus, resulting in high expression of CHOP and other ER chaperones (mainly GRP78). As a transcription regulator, CHOP promotes the expression of fibronectin and vimentin, which in turn cause the formation of renal interstitial fibrosis.

Article Snippet: The primary antibodies used were: polyclonal sheep anti-human UMOD antibody (R&D Systems, USA; Cat #AF6144, 1:100), proximal tubule brush border labeling antibody LTL-488 (Fluorescein Lotus Lectin, Vector Laboratories, USA, FL-1321; 1:1000), anti-GRP78 BiP antibody (abcam, Cambridge, UK; ab21685; 1:500) and CHOP (L63F7) mouse mAb (Cell Signaling Technology, Danvers, MA, USA; #2895; 1:50).

Techniques: Synthesized, Membrane, Mutagenesis, Expressing

Journal: PLoS ONE

Article Title: C-Type Lectin Receptor Dectin-2 Binds to an Endogenous Protein β-Glucuronidase on Dendritic Cells

doi: 10.1371/journal.pone.0169562

Figure Lengend Snippet: (A and B) Identification of Dectin-2-binding molecules on BMDCs. Affinity-purified proteins using Ig, Dectin-2 QPD -Ig (QPD) and Dectin-2-Ig (WT) were eluted with (A) heating or (B) addition of elution buffer containing 100mM Mannose. Top 5 proteins with high Mascot score were listed on the right side of the pictures. (C, D) Gusb expressions in BMDCs. Affinity-precipitated fractions with Ig and Dectin-2-Ig (D2) from whole cell lysate (WCL) of BMDCs (C) or culture supernatants (D) were analyzed by western blotting using anti-Gusb or anti-hIg mAb. (E) Reporter cells expressing FcRγ together with Dectin-2 were incubated with indicated cell numbers of Gusb-overexpressing RAW264.7 cells (black circle) or parental cells (Grey circle). (F) Dectin-2 reporter cells were incubated with Gusb-overexpressing RAW264.7 cells in the presence of mannose (100, 80, 60, 40 mM from the right) for 18 h. These data are presented as the means ± S.D. for duplicate assays, and representative of at least two independent experiments.

Article Snippet: Polyclonal anti-Gusb Ab was from proteintech.

Techniques: Binding Assay, Affinity Purification, Western Blot, Expressing, Incubation

Journal: PLoS ONE

Article Title: C-Type Lectin Receptor Dectin-2 Binds to an Endogenous Protein β-Glucuronidase on Dendritic Cells

doi: 10.1371/journal.pone.0169562

Figure Lengend Snippet: (A) Schematic diagram of N-glycosylation sites on Gusb. (B) The importance of glycosylation for binding of Dectin-2. Flag-tagged Gusb were immunoprecipitated with biotinylated anti-Flag (M2) antibody followed by enrichment with streptavidin beads. After incubation of Flag-tagged Gusb proteins with Dectin-2-Ig, co-precipitated Dectin-2-Ig was detected by western blotting using anti-hIg mAb.

Article Snippet: Polyclonal anti-Gusb Ab was from proteintech.

Techniques: Glycoproteomics, Binding Assay, Immunoprecipitation, Incubation, Western Blot

Journal: PLoS ONE

Article Title: C-Type Lectin Receptor Dectin-2 Binds to an Endogenous Protein β-Glucuronidase on Dendritic Cells

doi: 10.1371/journal.pone.0169562

Figure Lengend Snippet: (A) Gusb –/– mice were established by CRISPR-Cas9 system. Expression of Gusb on BMDCs was analyzed by western blotting. (B) Dectin-2-reporter cells recognized Gusb-deficient BMDCs. Reporter cells expressing Dectin-2 together with FcRγ were incubated with WT (black bar) or Gusb-deficient (grey bar) BMDCs for 18 h.

Article Snippet: Polyclonal anti-Gusb Ab was from proteintech.

Techniques: CRISPR, Expressing, Western Blot, Incubation