Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Location of missense and nonsense mutations in SLC9A6/NHE6 variants of patients with Christianson syndrome. A, schematic planar drawing of the predicted membrane topology of the longest splice-variant of mammalian NHE6 and location of the mutations (yellow circles). Two consensus N-linked glycosylation sites (128NVT and 145NVS) within extracellular loop 2 have been verified experimentally (data not shown) and are illustrated in the drawing. B, phylogenetic comparison of the primary sequence of segments containing the various mutations in NHE6. The affected residues are shaded in black.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Membrane, Variant Assay, Glycoproteomics, Comparison, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Structure homology modeling of transmembrane NHE6 variants associated with Christianson syndrome. A, front (membrane aspect, left panel) and top (extracellular aspect, right panel) views of a 3D structure homology model of dimeric human NHE6 based on the crystal structure of the bacterial T. thermophilus Na+/H+ antiporter NapA (TtNapA) (Protein Data Bank accession code 5bz3; 2.30 Å, 15% identity, 27% similarity), which provided the broadest coverage, highest resolution, and best spatial fit compared with other crystallized bacterial Na+/H+ antiporters. The proposed structure includes only the membrane-spanning helices (M2–M12; amino acids 74–540) that aligned with homologous segments of TtNapA. The top view includes the locations of the transmembrane-localized residues Leu-188 and Gly-383 mutated in CS. B, molecular dynamics simulation of structural changes predicted to occur in TM4 (highlighted in cyan) upon substitution of Leu-188 with Pro (L188P). The monomeric forms of NHE6 WT and L188P are illustrated. C and D, structural perturbations predicted to occur in the intramembranous re-entrant (R) loop between helices M8 and M9 upon substitution of Gly-383 with Asp (G383D). C, upper and lower panels show front and top views, respectively, of Gly-383, which is packed tightly against amino acids Phe-373, Ala-376, and Glu-377 (top left). Mutation of Gly-383 to Asp would result in steric clashes between these residues and interfere with the packing between these segments of the R-loop. D, G383D substitution would also disrupt interactions of R-loop residues Trp-379, Phe-381, and especially Thr-382 with residues Asp-92 and Ile-296 in helix M7 and residues Ile-330, Phe-331, and Ser-334 in helix M8.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Membrane, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the biosynthetic maturation of NHE6 variants. A, AP-1 cells transiently expressing NHE6GFP WT or CS-linked variants were lysed at the indicated time points (6–48 h) post-transfection. Equal amounts of proteins (20 μg) were subjected to SDS-PAGE and immunoblotting with a polyclonal anti-GFP antibody. NHE6 migrates as multiple bands: higher molecular weight bands represent the fully-glycosylated (fg) and core-glycosylated (cg) dimeric (d) forms of the exchanger that do not fully dissociate under SDS-PAGE conditions, whereas lower molecular weight bands represent fully-glycosylated and core-glycosylated forms of the dissociated monomeric (m) protein. The blots were stripped and reprobed with a mouse monoclonal anti-GAPDH antibody to control for protein loading. B, ratios of fully-glycosylated protein (monomer and dimer)/total NHE6 protein (fg/total) were quantified by densitometry of X-ray films exposed in the linear range and analyzed using ImageJ software. Values represent the mean ± S.D. of three different experiments. Significance was determined by two-way ANOVA with a post hoc Tukey test. The NHE6 variants clustered into two groups: 1) WT, A9S, and R568Q and 2) L188P, G383D, E547*, and W570*, with variants within each cluster yielding similar statistical values. Population means of the NHE6 variants are significantly different (F value = 26.4, p value = 1.5 × 10−16). Population means as a function of time are significantly different (F value = 48.6, p value = 1.9 × 10−19). § indicates significance (p < 0.01) of the means of NHE6 variants within a cluster relative to the 12-h time point. Asterisks indicate significance (★, p < 0.05, and ★★, p < 0.01) of the means between clusters of NHE6 variants at the indicated time points.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Expressing, Transfection, SDS Page, Western Blot, Molecular Weight, Control, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the protein stability of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT of CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide (CHX) for the indicated time points and lysed, and equal amounts of protein (20 μg) were analyzed by Western blotting using a mouse monoclonal anti-HA antibody. Blots were reprobed with a mouse monoclonal anti-GAPDH antibody to control for loading. Blots are representative images from four separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Effect of proteasomal and lysosomal inhibitors on cellular clearance of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT or CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide (CHX) for the indicated time points in the presence of diluent (DMSO) and the proteasomal inhibitor MG-132 (40 μm) or the lysosomal inhibitor leupeptin/pepstatin (LeuP, 100 μg/ml). Total-cell lysates were analyzed by Western blotting with a mouse monoclonal HA antibody. Membranes were also probed for β-tubulin expression as a loading control. The immunoblots are representative of three separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Effect of the lysosomotropic agent chloroquine on cellular clearance of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT or CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide for the indicated time points in the presence of diluent (H2O) or chloroquine (CQ, 500 μm). Total-cell lysates were analyzed by Western blotting with a mouse monoclonal HA antibody. Membranes were also probed for β-tubulin expression as a loading control. The immunoblots are representative of four separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Subcellular detection of NHE6 variants in recycling endosomes in transfected AP-1 cells. A, AP-1 cells were transiently transfected with mCherry fluorescent protein-tagged NHE6 (NHE6ChFP) WT or CS-linked variants. Forty eight hours post-transfection, cells were incubated with the recycling endosomal marker Alexa Fluor 488–conjugated transferrin (Tf-AF488, 10 μg/ml) for 45 min, fixed in 4% paraformaldehyde, mounted onto glass slides, and examined by confocal microscopy. Images show each channel individually, with merged images of the NHE6ChFP and Tf-AF488 channels. Scale bars represent 10 μm. B, quantitation of the degree of NHE6 overlapping with Tf-AF488 as determined by calculating the thresholded Mander's coefficient (M1) using ImageJ software and the JACoP plugin. Data are plotted as a box chart, with the central white square indicating the mean, the box representing the S.E., and the error bars showing the S.D. (n = 6–8 cells). Significance from WT was determined by one-way repeated measures ANOVA (F value = 6479.8, p value = 5.6 × 10−9), with a post hoc Dunnett's test, ★★★, p < 0.001.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Incubation, Marker, Confocal Microscopy, Quantitation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Subcellular localization of certain CS variants in the endoplasmic reticulum in transfected AP-1 cells. A, AP-1 cells were transiently transfected with mCherry fluorescent protein-tagged NHE6 (NHE6ChFP) WT or CS-linked variants. Forty eight hours post-transfection, cells were immunostained for endogenous CANX, fixed in 4% paraformaldehyde, mounted onto glass slides, and examined by confocal microscopy. Images show each channel individually, with merged images of the NHE6ChFP and CANX channels. Scale bars represent 10 μm. B, quantitation of the degree of NHE6 overlapping with CANX as determined by calculating the thresholded Mander's coefficient (M1) using ImageJ software and the JACoP plugin. Data are plotted as a box chart, with the central white square indicating the mean, the box representing the S.E., and the error bars showing the S.D. (n = 6–8 cells). Significance from WT was determined by one-way repeated measures ANOVA (F value = 3012.9, p value = 1.7 × 10−10), with a post hoc Dunnett's test, ★★★, p < 0.001.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Confocal Microscopy, Quantitation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the functional properties of NHE6 variants. A, biochemical determination of plasma membrane trafficking of NHE6GFP WT or CS-linked variants using a cell-surface biotinylation assay. Cell-surface proteins were labeled with N-hydroxysulfosuccinimidyl–SS–biotin in AP-1 cells expressing the NHE6GFP constructs after 48 h. Total-cell lysates (left panel; protein loading ranged from 10 to 50 μg of protein per sample as indicated below the blot) and biotinylated fractions (right panel; representing 20–100% of plasma membrane proteins extracted per sample) were examined by Western blotting with polyclonal anti-GFP and monoclonal anti-GAPDH antibodies. Representative blots from three experiments are shown. B and C, surface expression and endocytosis of external triple flag tag–labeled NHE6 (3FNHE6HA) constructs in transiently transfected (48 h) AP-1 cells using a cell-based ELISA. Mean intensity fluorescence (M.I.F.) units were determined as a function of the cellular protein concentration and then normalized as percentage (M.I.F. units for WT (100%): 25,100 ± 6,348, n = 4). The surface expression of each construct at time 0 min (before the start of internalization) is charted in B (n = 3–4 experiments). Significance from WT-expressing cells was determined using a one-way repeated measures ANOVA (F value = 463.3, p value = 0.0022), with a post hoc Dunnett's test; *, p < 0.05. Percentage internalization of NHE6 constructs normalized to the zero time point are presented in C and represent the mean ± S.D. (n = 3–4 experiments). The NHE6 variants clustered into two groups: 1) WT, A9S, and R568Q, and 2) L188P, G383D, E547*, and W570*, with variants within each cluster yielding similar statistical values. Significance from WT cells at the 5- and 15-min time points was determined using a one-way ANOVA (F value = 9.43, p value = 4.48 × 10−5), with a post hoc Tukey test; ★, p < 0.05. D, transferrin uptake in HeLa cells transiently transfected (48 h) with GFP or NHE6GFP constructs. The initial uptake (5 min) of Alexa 633–conjugated transferrin (Tf-AF633) was measured in 1 × 104 GFP-positive HeLa cells per experiment by flow cytometry (M.I.F. units for GFP control: 10,204 ± 1554, n = 4). Data were normalized as a percentage and displayed as percent change from GFP control cells. Significance from control cells was determined using a one-way repeated measures ANOVA (F value = 320.7, p value = 3.8 × 10−4), with a post hoc Dunnett's test; ★★, p < 0.001. E, recycling endosomal pH (pHe) was measured in AP-1 cells in the absence or presence of transiently transfected (48 h) NHE6ChFP constructs by fluorescence ratio image analysis of the internalized pH-sensitive probe FITC-conjugated human transferrin (Tf–FITC). Data represent the average endosomal pHe per cell pooled from three separate experiments (8–12 cells per construct/experiment; n = 24–36). Significance was determined by one-way ANOVA (F value = 40.02, p value = 0), with a post-hoc Tukey test; ★★, p < 0.001. Data in B, D, and E are plotted as box charts, with the central white square indicating the mean; the box representing the S.E.; and the error bars showing the S.D. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Functional Assay, Clinical Proteomics, Membrane, Cell Surface Biotinylation Assay, Labeling, Expressing, Construct, Western Blot, FLAG-tag, Transfection, In-Cell ELISA, Fluorescence, Protein Concentration, Flow Cytometry, Control

Journal: Nature Communications

Article Title: Reversal of pancreatic desmoplasia by re-educating stellate cells with a tumour microenvironment-activated nanosystem

doi: 10.1038/s41467-018-05906-x

Figure Lengend Snippet: Reversal of activated PSCs and ECM reduction in vitro. a Immunofluorescence (IF) staining of HSP47, nile red staining of lipid droplets, IF staining of α-SMA, Sirius red staining of deposited collagen and IF staining of fibronectin in PSCs after treatment with the indicated formulations for 48 h at pH 6.5. Scale bars, 20 μm. b Quantification of the normalised HSP47 protein expression (using Image J software). c Quantification of the normalised α-SMA protein expression (using Image J software). d Normalised deposited collagen by measuring extracted Sirius red dye at 540 nm. e Quantification of the normalised fibronectin protein expression (using Image J software). f Western blot analysis of secreted collagen I in the PSCs culture supernatant and fibronectin in whole cell lysates after treatment with the indicated formulations for 48 h at pH 6.5. g Quantitative analysis of the normalised fibronectin and collagen I protein expression (using Image J software). The data are shown as the mean ± s.d. ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test)

Article Snippet: Rabbit polyclonal antibodies against GAPDH (cat# 10494-1-AP), heat shock protein 47 (HSP47, cat# 10875-1-AP), glial fibrillary acidic protein (GFAP, cat# 16825-1-AP), fibronectin (cat# 15613-1-AP), albumin (16475-1-AP), collagen type I (cat# 14695-1-AP) and cytokeratin 19 (cat# 10712-1-AP) were obtained from Proteintech (Chicago, USA).

Techniques: In Vitro, Immunofluorescence, Staining, Expressing, Software, Western Blot

Journal: Nature Communications

Article Title: Reversal of pancreatic desmoplasia by re-educating stellate cells with a tumour microenvironment-activated nanosystem

doi: 10.1038/s41467-018-05906-x

Figure Lengend Snippet: Penetration of small molecules in Panc-1/PSC stroma-rich spheroids (PDAC-SS). PDAC-SS was generated by hanging drop culture of PSCs (pre-treated with different formulations) and Panc-1 cells. a Representative bright field images, Sirius red staining images, fibronectin immunofluorescence images and penetrated Hoechst 33342 fluorescence images of PDAC-SS. Scale bars, 100 μm. b – d Quantification of the normalised collagen positive (Sirius red stained) area ( b ), fibronectin protein expression ( c ) and penetration depth of Hoechst 33342 (d; using Image J software). Hoechst 33342, a low-molecular weight fluorescent DNA-binding dye, was used as a probe to visualise the penetrability of PDAC-SS. The data are shown as the mean ± s.d. ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test)

Article Snippet: Rabbit polyclonal antibodies against GAPDH (cat# 10494-1-AP), heat shock protein 47 (HSP47, cat# 10875-1-AP), glial fibrillary acidic protein (GFAP, cat# 16825-1-AP), fibronectin (cat# 15613-1-AP), albumin (16475-1-AP), collagen type I (cat# 14695-1-AP) and cytokeratin 19 (cat# 10712-1-AP) were obtained from Proteintech (Chicago, USA).

Techniques: Generated, Staining, Immunofluorescence, Fluorescence, Expressing, Software, Molecular Weight, Binding Assay

Journal: Nature Communications

Article Title: Reversal of pancreatic desmoplasia by re-educating stellate cells with a tumour microenvironment-activated nanosystem

doi: 10.1038/s41467-018-05906-x

Figure Lengend Snippet: Stroma modulation in Panc-1/PSC subcutaneous xenografts. a Scheme of different treatment formulations for stroma modulation. The desmoplastic pancreatic subcutaneous xenografts were established by co-inoculation of Panc-1 and PSCs in BALB/c nude mice. Various formulations (ATRA: 2.4 mg/kg; siRNA: 0.97 mg/kg) were given intravenously every 2 days for three injections. b Body weight changes of mice during treatment. c Tumour growth curves during treatment. d Western blot analysis of HSP47 protein in tumours. e Quantitative analysis of the normalised HSP47 protein expression in tumours (using Image J software). f Histological studies with H&E, trichrome staining of collagen and immunohistochemical staining of HSP47 and fibronectin in tumour sections. Scale bars, 50 μm. g – i Quantitative analysis of the normalised HSP47 ( g ), collagen ( h ) and fibronectin ( i ) protein levels (using Image J software). The data are shown as the mean ± s.d. ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test)

Article Snippet: Rabbit polyclonal antibodies against GAPDH (cat# 10494-1-AP), heat shock protein 47 (HSP47, cat# 10875-1-AP), glial fibrillary acidic protein (GFAP, cat# 16825-1-AP), fibronectin (cat# 15613-1-AP), albumin (16475-1-AP), collagen type I (cat# 14695-1-AP) and cytokeratin 19 (cat# 10712-1-AP) were obtained from Proteintech (Chicago, USA).

Techniques: Western Blot, Expressing, Software, Staining, Immunohistochemical staining

Journal: Molecular Neurodegeneration

Article Title: A Christianson syndrome-linked deletion mutation (∆ 287 ES 288 ) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death

doi: 10.1186/s13024-016-0129-9

Figure Lengend Snippet: Expression and post-translational processing of NHE6 ΔES mutant protein is impaired in transfected AP-1 and SH-SY5Y cells. a Schematic drawing of the predicted membrane topology of mammalian NHE6v1 (based on sequence alignment and transmembrane organization of NHE1 proposed by Wakabayashi et al. and Nygaard et al. ) and locations of mutations ( red shading ) identified by Gilfillan et al. in patients with Christianson syndrome. The blue shading in the second extracellular loop (EL2) represents the additional 32 amino acids (residues 145–176) present in the NHE6v1 splice-variant. Two predicted N -glycosylation sites within EL2 are also illustrated. b AP-1 and c , SH-SY5Y cells were transiently transfected (24 h) with NHE6 HA WT or ΔES mutant. Total cell lysates of WT and ΔES-transfectants were examined by SDS-PAGE. The immunoblots were probed with a mouse monoclonal anti-HA antibody (α-HA m ) to detect NHE6v1. NHE6v1 migrates as multiple bands: slower migrating high molecular weight bands representing the fully-glycosylated ( fg ) and core-glycosylated ( cg ) dimeric forms of the exchanger (~200 and 175 kDa, respectively) that do not fully dissociate under SDS-PAGE conditions and faster migrating fully-glycosylated ( fg , ~100 kDa) and core-glycosylated ( cg , ~70 kDa) and unglycosylated ( ug , ~65 kDa) forms of the monomeric protein. To control for protein loading, the blots were reprobed with a mouse monoclonal anti-GAPDH antibody (α-GAPDH m ). d , e To confirm the nature of the oligosaccharide modifications of the NHE6 bands, AP-1 cells transiently transfected (24 h) with WT or ΔES constructs were lysed in non-detergent buffers and post-nuclear supernatants were left untreated or incubated with either endoglycosidase H (EndoH), which cleaves only asparagine-linked mannose-rich oligosaccharides (i.e., core-glycosylated) but not more highly processed complex oligosaccharides (i.e., fully-glycosylated), or peptide-N-glycosidase F (PNGaseF) which cleaves between the innermost N-acetylglucosamine and asparagine residues of all oligosaccharide structures (i.e., high mannose, hybrid, and complex). The lysates were then subjected to SDS-PAGE and immunoblotting with an α-HA m antibody. The data are representative of three independent experiments

Article Snippet: After removing a small fraction of the pre-cleared cell lysate for immunoblotting, the remaining lysate was divided into two equal fractions: one fraction was used to immunoprecipitate the NHE6 protein overnight at 4 °C with a rabbit polyclonal anti-HA antibody (Abcam); the second fraction was subjected to immunoprecipitation with a nonspecific rabbit IgG antibody (Southern Biotech) as a negative control.

Techniques: Expressing, Mutagenesis, Transfection, Membrane, Sequencing, Variant Assay, Glycoproteomics, SDS Page, Western Blot, High Molecular Weight, Control, Construct, Incubation

Journal: Molecular Neurodegeneration

Article Title: A Christianson syndrome-linked deletion mutation (∆ 287 ES 288 ) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death

doi: 10.1186/s13024-016-0129-9

Figure Lengend Snippet: Biosynthetic maturation of NHE6 is reduced for the ∆ES mutant. AP-1 cells were transiently transfected with a NHE6v1 HA WT or b ΔES and lysed at the indicated time points over a 48 h period. Equal amounts of proteins were subjected to SDS-PAGE and immunoblotting with a monoclonal anti-HA antibody (α-HA). The identities of the various NHE6 bands are as described in the legend to Fig. . For the ΔES immunoblot in panel B , a longer X-ray film exposure (18X) of the 36 h and 48 h time points is also shown. The same immunoblots were also probed with a monoclonal anti-β-tubulin antibody as a loading control. c-d Densitometric quantification of the relative abundances of the monomeric and dimeric forms of WT or ΔES was assessed using ImageJ software and expressed as ratios of fully glycosylated/total protein (fg/total). For quantification, multiple exposures of the immunoblots were taken to ensure the signal intensities of the bands were within the linear range of the X-ray film. Data are shown as mean ± standard error of the mean (S.E.M.) of four different experiments

Article Snippet: After removing a small fraction of the pre-cleared cell lysate for immunoblotting, the remaining lysate was divided into two equal fractions: one fraction was used to immunoprecipitate the NHE6 protein overnight at 4 °C with a rabbit polyclonal anti-HA antibody (Abcam); the second fraction was subjected to immunoprecipitation with a nonspecific rabbit IgG antibody (Southern Biotech) as a negative control.

Techniques: Mutagenesis, Transfection, SDS Page, Western Blot, Control, Software

Journal: Molecular Neurodegeneration

Article Title: A Christianson syndrome-linked deletion mutation (∆ 287 ES 288 ) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death

doi: 10.1186/s13024-016-0129-9

Figure Lengend Snippet: Stability of NHE6 is diminished for ΔES mutant. a AP-1 cells were transiently transfected with NHE6v1 HA WT or ΔES mutant for 24 h and then treated with 150 μg/mL cycloheximide for the indicated time points, lysed and analysed by SDS-PAGE and immunoblotting with a mouse monoclonal anti-HA (α-HA m ) antibody. Equal amounts of proteins were loaded, as shown by probing the membranes with a monoclonal anti-GAPDH antibody (α-GAPDH m ). b Quantitative analysis by densitometry of NHE6v1 WT and ΔES protein abundance (normalized to GAPDH levels) as a function of time in the presence of cycloheximide. Values represent the mean ± S.E.M. of three separate experiments. c-d AP-1 cells were transiently transfected with NHE6v1 HA WT ( c ) or ΔES ( d ) for 24 h and then treated with 150 μg/mL cycloheximide for the indicated time points in the presence of DMSO (vehicle), the proteasomal inhibitors MG-132 (40 μM) or lactacystin (LC, 30 μM) ( left panels ), or the lysosomal inhibitors leupeptin/pepstatin (LeuP, 100 μg/ml) or chloroquine (CQ, 500 μM) ( right panels ). Cellular lysates were analysed by immunoblotting with a mouse monoclonal α-HA m antibody. Membranes were also probed with a mouse monoclonal α-GAPDH m antibody as a loading control

Article Snippet: After removing a small fraction of the pre-cleared cell lysate for immunoblotting, the remaining lysate was divided into two equal fractions: one fraction was used to immunoprecipitate the NHE6 protein overnight at 4 °C with a rabbit polyclonal anti-HA antibody (Abcam); the second fraction was subjected to immunoprecipitation with a nonspecific rabbit IgG antibody (Southern Biotech) as a negative control.

Techniques: Mutagenesis, Transfection, SDS Page, Western Blot, Quantitative Proteomics, Control

Journal: Molecular Neurodegeneration

Article Title: A Christianson syndrome-linked deletion mutation (∆ 287 ES 288 ) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death

doi: 10.1186/s13024-016-0129-9

Figure Lengend Snippet: Ubiquitination of NHE6 is increased for the ΔES mutant. a AP-1 cells transiently expressing NHE6v1 HA WT or ΔES for 24 h were lysed and total levels of NHE6 were analyzed by immunoblotting with a mouse monoclonal anti-HA (α-HA m ) antibody. GAPDH was measured as a loading control ( panel 1, far left ). The level of ubiquitinated proteins in the total cell lysates (TCL) was examined using a mouse monoclonal anti-ubiquitin (α-Ub m ) antibody ( panel 2 ). The TCL were subjected to immunoprecipitation with a non-specific rabbit polyclonal IgG (α-IgG p ) antibody ( panel 3 ) or a rabbit polyclonal anti-HA (α-HA p ) antibody ( panel 4 ) and analyzed with a mouse monoclonal α-Ub m antibody to detect the ubiquitination levels of NHE6 WT versus ΔES. The membrane from panel 4 was then stripped and reprobed with a mouse monoclonal α-HA m antibody to examine the total amount of NHE6 WT and ΔES retrieved by immunoprecipitation ( panel 5 ). b Quantitative analysis by densitometry of the ratio of ubiquitinated to total protein abundance of NHE6v1 WT and ΔES in the immunopreciptated pellets. Values represent the mean ± S.E.M. of three separate experiments. Statistical significance was assessed using a Student’s t -test, * p < 0.01

Article Snippet: After removing a small fraction of the pre-cleared cell lysate for immunoblotting, the remaining lysate was divided into two equal fractions: one fraction was used to immunoprecipitate the NHE6 protein overnight at 4 °C with a rabbit polyclonal anti-HA antibody (Abcam); the second fraction was subjected to immunoprecipitation with a nonspecific rabbit IgG antibody (Southern Biotech) as a negative control.

Techniques: Ubiquitin Proteomics, Mutagenesis, Expressing, Western Blot, Control, Immunoprecipitation, Membrane, Quantitative Proteomics

Journal: Molecular Neurodegeneration

Article Title: A Christianson syndrome-linked deletion mutation (∆ 287 ES 288 ) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death

doi: 10.1186/s13024-016-0129-9

Figure Lengend Snippet: Subcellular distribution of NHE6 ΔES is altered in AP-1 cells. a Plasma membrane location of NHE6v1 as measured biochemically using a cell-surface biotinylation assay. To detect cell surface expression of NHE6v1, a triple Flag epitope-tag was inserted into the first predicted extracellular loop of NHE6v1 HA ( 3F NHE6v1 HA ), as illustrated in the upper panel . AP-1 cells were transiently transfected with 3F NHE6v1 HA WT or ΔES for 36 h and cell surface proteins were labeled with biotin as described in ‘Material and Methods’. Total cell lysates (TCL) were prepared and a small portion representing the total fraction was removed. The remaining supernatants containing equal amounts of total protein for WT and ΔES were loaded onto NeutrAvidin® Agarose beads to purify the biotinylated cell surface proteins from the non-biotinylated (intracellular) proteins. For the TCL and the remaining non-biotinylated fractions, aliquots containing 20 μg and 80 μg protein for WT and ΔES, respectively, were examined by Western blotting ( left and right lower panels, respectively ). For the plasma membrane fraction, 25 % and 100 % of the biotinylated proteins extracted from the total cell lysates of WT and ΔES transfectants, respectively, were subjected to Western blotting ( middle lower panel ). All immunoblots were probed with mouse monoclonal anti-HA m antibody to detect NHE6 and anti-GAPDH m antibody to assess the enrichment of the biotinylated fraction, as GAPDH is a cytosolic protein. b Confocal fluorescence microscopy and transmitted light images of fixed non-permeabilized AP-1 cells showing surface expression of 3F NHE6v1 HA WT or ΔES. Scale bars represent 5 μM. c AP-1 cells were transiently transfected with 3F NHE6v1 HA WT ( upper panels ) or ΔES ( lower panels ). Thirty-six h after transfection, cells were loaded with Alexa Fluor 594 -labelled transferrin (Tf-AF 594 , 10 μg/mL) for 45 min, fixed in 4 % paraformaldehyde, permeabilized, mounted onto glass slides and then examined by confocal microscopy. Footprints of the transfected cells are indicated as white dotted outlines and were derived from the transmitted light images ( far left panels ). Scale bars represent 10 μm

Article Snippet: After removing a small fraction of the pre-cleared cell lysate for immunoblotting, the remaining lysate was divided into two equal fractions: one fraction was used to immunoprecipitate the NHE6 protein overnight at 4 °C with a rabbit polyclonal anti-HA antibody (Abcam); the second fraction was subjected to immunoprecipitation with a nonspecific rabbit IgG antibody (Southern Biotech) as a negative control.

Techniques: Clinical Proteomics, Membrane, Cell Surface Biotinylation Assay, Expressing, FLAG-tag, Transfection, Labeling, Western Blot, Fluorescence, Microscopy, Confocal Microscopy, Derivative Assay

Journal: Molecular Neurodegeneration

Article Title: A Christianson syndrome-linked deletion mutation (∆ 287 ES 288 ) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death

doi: 10.1186/s13024-016-0129-9

Figure Lengend Snippet: Uptake of transferrin is impaired in HeLa cells expressing NHE6 ΔES. a Comparison of uptake of transferrin-Alexa Fluor 633 (Tf-AF 633 ; 10 μg/ml, 5 min) in AP-1 vs. HeLa cells. Significance was measured using a one-sample Student’s t -test; * p < 0.05. b Surface expression of transferrin receptor (TfR) in AP-1 vs. HeLa cells was measured by cell surface biotinylation. A representative immunoblot shows TfR and GAPDH expression in AP-1 and HeLa cells ( left panel ). Quantification of surface and total TfR relative to total GAPDH expression from three different experiments; values are normalized to the TfR/GAPDH ratio in AP-1 cells ( right panel ). c Transient expression of GFP, NHE6v1 GFP WT or ΔES in HeLa cells after 48 h. Representative immunoblot probed with a polyclonal anti-GFP (α-GFP P ) antibody. d Uptake of Tf-AF 633 was monitored in HeLa cells expressing GFP, NHE6v1 GFP WT or ΔES. Median fluorescence intensity (M.I.F.) of Tf-AF 633 was measured in 10 4 GFP-positive cells by flow cytometry. Data were normalized and represent mean ± S.E.M. of eight different experiments. Significance was established using a one-way ANOVA followed by a Tukey test, ** p < 0.01. e Uptake of Tf-AF 633 in HeLa cells expressing GFP, NHE6v1 GFP WT or ΔES in the absence (−) or presence (+) of 10-fold excess unlabeled Tf. The M.I.F. of Tf-AF 633 was measured in 10 4 GFP-positive cells by flow cytometry. Data represent mean ± S.E.M. of three different experiments, ** p < 0.01. f qPCR of NHE6 mRNA levels in HeLa cells treated for 72 h with non-target (scrambled) siRNA or NHE6 siRNA. g Uptake of Tf-AF 633 was monitored in HeLa cells expressing non-target siRNA or NHE6 siRNA for 72 h. The M.I.F. of Tf-AF 633 was measured in 10 5 cells by flow cytometry. Data were normalized and represent mean ± S.E.M. of three different experiments. Significance was established using a one-sample Student’s t -test, ** p < 0.01. h Cell surface levels of TfR in HeLa cells transiently expressing GFP, NHE6v1 GFP WT or ΔES were determined by cell surface biotinylation. Representative immunoblot of TfR expression at the cell surface and the total fraction is shown in the left panel . Quantification by densitometry of cell surface/total TfR levels from three different experiments is shown in the right panel . Values were normalized to TfR amounts present in GFP-expressing cells and represent the mean ± S.E.M. Significance was established using a one-sample Student t -test, ** p <0.01, * p <0.05

Article Snippet: After removing a small fraction of the pre-cleared cell lysate for immunoblotting, the remaining lysate was divided into two equal fractions: one fraction was used to immunoprecipitate the NHE6 protein overnight at 4 °C with a rabbit polyclonal anti-HA antibody (Abcam); the second fraction was subjected to immunoprecipitation with a nonspecific rabbit IgG antibody (Southern Biotech) as a negative control.

Techniques: Expressing, Comparison, Western Blot, Fluorescence, Flow Cytometry

Journal: Molecular Neurodegeneration

Article Title: A Christianson syndrome-linked deletion mutation (∆ 287 ES 288 ) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death

doi: 10.1186/s13024-016-0129-9

Figure Lengend Snippet: Expression of NHE6 ΔES enhances apoptosis in AP-1 cells. a Confocal microscopy of fixed AP-1 cells expressing 3F NHE6v1 HA WT ( upper panels ) or ΔES ( lower panels ). NHE6 was labelled with a mouse monoclonal anti-HA m antibody and an Alexa Fluor 488 -conjugated goat anti-mouse secondary antibody. Actin filaments were labelled with rhodamine-phalloidin and the nuclei were stained with DAPI. b Transient expression (48 h) of GFP, NHE6v1 GFP WT or ΔES in AP-1 cells. Representative immunoblot probed with a polyclonal anti-GFP antibody (α-GFP P ). c Flow cytometry analysis of AP-1 cells transfected with GFP alone, NHE6 GFP WT or ΔES. Forty-eight hours after transfection, cells were labeled with Annexin V-APC and propidium iodide (PI) and 10 4 GFP-positive cells were examined by flow cytometry for each transfectant. Annexin V- and PI- double negative cells represent viable cells. Cells taking up only PI (PI+) are indicative of dead cells; Annexin V+ positive cells indicate early apoptotic cells whereas Annexin V+ and PI+ double positive cells represent late apoptotic cells. Results are shown as mean ± S.E.M. of eight independent experiments. Significance was determined using a paired two-tailed Student t -test, ** p < 0.01. d AP-1 cells transiently expressing GFP alone, NHE6 GFP WT or ΔES were isolated by cell sorting and then assayed for caspase 3/7 activity as described in “Materials and Methods”. Data were normalized to values for GFP-expressing cells and displayed as mean ± S.E.M. of four independent experiments, each done in triplicate. Significance was determined using a paired two-tailed Student t -test, * p < 0.05

Article Snippet: After removing a small fraction of the pre-cleared cell lysate for immunoblotting, the remaining lysate was divided into two equal fractions: one fraction was used to immunoprecipitate the NHE6 protein overnight at 4 °C with a rabbit polyclonal anti-HA antibody (Abcam); the second fraction was subjected to immunoprecipitation with a nonspecific rabbit IgG antibody (Southern Biotech) as a negative control.

Techniques: Expressing, Confocal Microscopy, Staining, Western Blot, Flow Cytometry, Transfection, Labeling, Two Tailed Test, Isolation, FACS, Activity Assay

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: (A) Experimental scheme. Thirty-two-cell Xenopus embryos were co-injected with 5 nl of control (Co) or FGFR1 morpholino (MO), 15 ng each, along with GFP RNA (90 pg) as a lineage tracer into one dorsal animal blastomere. Embryos were collected at stage 15 (st.15) and co-immunostained for Vangl2 (red) and GFP (green). The anteroposterior (AP) axis is indicated. Representative en face neural plate images are shown in (B) and (C), white box areas are magnified on the right to show merged (green+red) and single (red) channel images. (B) Control neuroectoderm with anteriorly polarized Vangl2 (arrows). (C) FGFR1MO-injected neuroectoderm lacking anterior Vangl2 accumulation (asterisks). (D) Frequencies of GFP-positive cells with anteriorly enriched Vangl2 in the CoMO and FGFR1MO injected neuroectoderm. Numbers of scored cells (n) are indicated on the top of each bar. Scoring was performed on 50-90 cells per embryo, three embryos per group. Data represent four independent experiments; *** p<0.001, two-tailed unpaired Student’s t test. Scale bar, 30 µm.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Representative image of neural plate, stage 15 of an embryo injected with FGFR1 morpholino (MO) and GFP RNA, and co-immunostained for Vangl2 and GFP as described in legend and Methods. A cell was scored as polarized if the fluorescence intensity at an anteroposterior (A-P) junction was at least twofold higher compared to the fluorescence intensity at the mediolateral (M-L) junction. Examples of a planar-polarized cell (arrow) and a non-polarized cell depleted of FGFR1 (asterisk) are shown. Merged channels (Vangl2 (red) +GFP(green)) are shown. A-P and M-L junctions are indicated by dashed boxes. Anteroposterior (A-P) axis is marked. Scale bar: 50 µm.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Fluorescence

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: (A) Experimental scheme. Thirty-two-cell stage Xenopus embryos were injected with RNAs encoding GFP (200 pg) and dominant-interfering FGFR1 construct (XFD, 400 pg) into one dorsal animal blastomere. Embryos were collected at stage 15 and co-immunostained for Vangl2 (red) and GFP (green). The dashed boxed area is magnified in (B-C’). The anteroposterior (A-P) axis is indicated. (B-B’) Representative images of neural plate cells expressing GFP tracer with anteriorly enriched Vangl2 (arrows). (C-C’) Vangl2 is not restricted to anterior cell boundaries in cells expressing XFD (asterisks). (D) Quantification of the mean ± s.d. frequencies of cells with anteriorly enriched Vangl2. Numbers of scored cells (n) are indicated on top of each bar. Three embryos were scored for each group (40-100 cells per embryo). Data represent three independent experiments. Statistical significance was assessed using a two-tailed Student’s t -test (***, p<0.001). Scale bar: 30 µm.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Construct, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Thirty-two-cell stage Xenopus embryos were injected into one dorsal animal blastomere with RNAs encoding GFP-Pk3 (150 pg), HA-Vangl2 (20 pg), and myristoylated BFP (BFP, 80 pg), with or without the dominant-interfering FGFR1 construct (XFD, 400 pg), and fixed at stage 14 (st.14). GFP-Pk3 (Pk3, green) and BFP (blue) fluorescence channels are shown. GFP-Pk3 (A, B) and merged GFP-Pk3+BFP fluorescence (A’, B’) is shown. Anteroposterior (A-P) axis is indicated. Dorsal view of representative images of neural plate cells expressing GFP-Pk3 without (A-A’, arrows) and with XFD (B-B’, asterisks). Cell membranes are marked by BFP (A’, B’). Scale bar: 30 µm. (C, D) GFP-Pk3 fluorescence around cell boundaries in control (C) and XFD-expressing (D) mosaic neuroepithelial cells was quantified as described in legend and Methods and plotted as a function of angle. In control cells, GFP-Pk3 fluorescence is enriched at the anterior side (90°), whereas it is evenly distributed in cells co-expressing XFD. Cell numbers (n) are shown.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Construct, Fluorescence, Expressing, Control

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: (A-C) FGFR signaling inhibition decreases Vangl2 tyrosine phosphorylation in Xenopus embryos. (A) experimental scheme for (B-C). Ectoderm explants were dissected from stage 9 embryos and used for pull down with Vangl2 antibody. (B) Explants were dissected from embryos injected with HA-Vangl2 RNA (40 pg), with or without dominant-interfering FGFR1 (XFD) and cultured until stage 12. (C) Reduction of endogenous Vangl2 tyrosine phosphorylation in ectoderm explants (stage 16) depleted of FGFR1 with FGFR1 MO (20 ng). (D-F) Vangl2 tyrosine phosphorylation depends on FGFR signaling activity in mouse embryonic stem (ES) cells. (D) Experimental scheme. (E, F) Lysates from wild-type and FGFR1/2 double knockout mouse ES cells were precipitated with anti-Vangl2 (E) or anti-phosphotyrosine (pY) (F) antibodies and immunoblotted as indicated. Cyclin B1 is a negative control (F).

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Inhibition, Phospho-proteomics, Injection, Cell Culture, Activity Assay, Double Knockout, Negative Control

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Xenopus embryos were injected with RNAs for HA-Vangl2 (40 pg) with or without XFD-3xFLAG (150 pg) and cultured until stage 12. Vangl2 tyrosine phosphorylation was analyzed by HA-trap pulldown followed by immunoblotting (IB) with anti-pY, anti-HA, or anti-FLAG antibodies as indicated.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Cell Culture, Phospho-proteomics, Western Blot

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Left: Experimental scheme. Ectoderm explants were dissected from Xenopus embryos injected with HA-Vangl2 RNA (40 pg) at stage 9 and cultured in the presence of FGF2 (25–100 ng/ml), with or without SU5402 (100 µM), until stage 12. Vangl2 phosphorylation was analyzed in anti-Vangl2 immunoprecipitates immunoblotted with anti-phosphotyrosine (pY) and anti-HA antibodies. Intensity ratios of pY to HA signals for control and FGF2-treated explants are shown.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Cell Culture, Phospho-proteomics, Control

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Four- to eight-cell Xenopus embryos were injected with RNAs for GFP-Vangl2, PTK7-GFP and FGFR1-FLAG (100 pg each) (A) or RNAs for HA-Vangl2 (50 pg) and either FGFR1-FLAG (40 pg) or Frizzled3-FLAG (400 pg) (B), lysed at stage 12 and subjected to immunoprecipitation (IP) with anti-FLAG to assess FGFR1-Vangl2 binding (A) or anti-Vangl2 to assess Vangl2 tyrosine phosphorylation (B). PTK7-GFP is a negative control in (A). Frizzled3-dependent Vangl2 band shift in (B) reflects S/T phosphorylation . An irrelevant portion of the membrane was removed. (C) FGF8 induces Vangl2 phosphorylation in Xenopus embryos. Embryos were injected with HA-Vangl2 with or without FGF8 plasmid DNAs (50 pg) each, lysed at stage 14 and precipitated with HA-trap followed by immunoblot with anti-pY antibodies.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Immunoprecipitation, Binding Assay, Phospho-proteomics, Negative Control, Electrophoretic Mobility Shift Assay, Membrane, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Formation of the complex between Vangl2 and FGFR1 was assessed in C17.2 neural progenitor cells (A) and HEK293T (B) cells. Cell lysates were precipitated with 1 µg of anti-Vangl2 antibody or control rabbit IgG. The immunoprecipitates and input lysates were probed with FGFR1 and Vangl2 antibodies. The band corresponding to the upper FGFR1 band in C17.2 and HEK293T cell lysates is detected in Vangl2 pulldowns from both cell lines (A, B; arrows). Molecular weight markers are indicated.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Control, Molecular Weight

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: (A) Xenopus embryos were injected with RNAs for Xenopus HA-Vangl2 (40 pg) with or without mouse FGFR1 and FGFR2 (40 pg each), or (B) DNA constructs encoding HA-Vangl2 (25 pg) with or without human FGFR1, FGFR1 Y372>C, or FGFR4 (50 pg each), (see Methods), and cultured until stage 12 (A) or stage 14 (B). Vangl2 tyrosine phosphorylation (Vangl2-pY) was analyzed by pull-downs with rabbit anti-HA antibody (A) or HA-trap beads (B), immunoblotted with anti-pY, anti-HA, or anti-FLAG antibodies as indicated. Expression of wild-type and mutated human FGFR constructs (B) was assessed with anti-phospho-Y653-FGFR1 antibody. Asterisk marks non-specific band in lysates (A, B).

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Construct, Cell Culture, Phospho-proteomics, Expressing

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Four- to eight-cell Xenopus embryos were injected into four animal blastomeres with GFP-Vangl2 RNA (40 pg), with or without FGFR1-FLAG RNA (10 pg or 20 pg). Ectoderm explants were dissected at stage 9, cultured until stage 12, lysed, and GFP-Vangl2 was pulled down using GFP-trap beads. Ectoderm explants were cultured with or without FGF2 (100 ng/mL) or SU5402 (100 µM). FGFR1 dose-dependently induced Vangl2 tyrosine phosphorylation. FGF2 strongly activated ERK (pERK) and weakly induced Vangl2 phosphorylation. Neither Vangl2 nor ERK phosphorylation was detected in SU5402-treated explants. Data represent three independent experiments.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Cell Culture, Phospho-proteomics

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Four-to-eight cell embryos were injected into four animal blastomeres with GFP-Vangl2 RNA, FGFR1-FLAG or FGFR1 FCPG -FLAG RNA (40 pg each), and were cultured until stage 12. GFP-Vangl2 was immunoprecipitated (IP) from embryo lysates using GFP-trap beads followed by immunoblotting (IB) with anti-pY, anti-GFP, anti-FLAG, anti-pERK1/2 and anti-ERK1/2 antibodies. FGFR1 FCPG containing mutations in the binding sites of known signaling mediators induced Vangl2 tyrosine phosphorylation but did not upregulate ERK1/2 activity.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Cell Culture, Immunoprecipitation, Western Blot, Binding Assay, Phospho-proteomics, Activity Assay

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: (A) Mapping major phospho-tyrosine (pY) sites in Vangl2. Embryos were injected with 40 pg of RNA encoding FGFR1 and the indicated HA-Vangl2 constructs. Vangl2 phosphorylation was analyzed in anti-HA pull-downs from stage 13 embryo lysates. Immunoblotting (IB) was performed using anti-pY, anti-HA or anti-FLAG antibodies, as indicated. One representative set of duplicate samples is shown. The graph below shows average pY/HA intensity ratios for the duplicates. (B) Alignment of N-terminal amino acid sequences of Vangl2 from several chordate species and Drosophila Van Gogh. The N-terminal tyrosine cluster (in red) is conserved in vertebrates but not in Drosophila . (C-E) Planar polarization of Vangl2 tyrosine phosphosite mutants in the neuroectoderm. (C) Experimental scheme. Two dorsal blastomeres of 16-cell embryos were coinjected with myrBFP RNA (80 pg) and RNAs encoding GFP-Pk3 (150 pg) HA-tagged Vangl2 (in D), Y7,10,12>F (Y>F, in E) or Y7,10,12>E (Y>E, in F) (20 pg each). Embryos were fixed at stages 14-15, and GFP and BFP fluorescence were imaged. Anterior enrichment of GFP-Pk3 is indicated by arrows (D-E’), while a non-polarized cell is marked by an asterisk (F-F’). The anteroposterior (AP) axis is indicated. (G) Quantification of GFP fluorescence for the GFP-Pk3-Vangl2 complexes in mosaically expressing cells is shown as a graph. Mean fluorescence is plotted along the cell circumference as a function of circular angle from 0 to 360 degrees relative to the AP axis, with fluorescence intensity shown in green (HA-Vangl2), pink (HA-Vangl2Y>F), and brown (HA-Vangl2Y>E) lines. The number of scored cells is indicated. The data are representative of four independent experiments.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Construct, Phospho-proteomics, Western Blot, Fluorescence, Expressing

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Embryos were injected with 40 pg of RNAs encoding FGFR1, FGFR1D623A and various HA-Vangl2 constructs as indicated. Vangl2 phosphorylation was analyzed in HA-trap immunoprecipitates from stage 12 embryo lysates. Immunoblotting (IB) was done with anti-pY, anti-HA or anti-FLAG antibodies as indicated.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Construct, Phospho-proteomics, Western Blot

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Four- to eight-cell Xenopus embryos were injected with RNAs encoding HA-Vangl2, Y7Y10Y12>F (Y>F), or Y7Y10Y12>E (Y>E) constructs (40 pg each). Vangl2 proteins were pulled down from stage 13 lysates using anti-HA antibody and probed with anti-pS14S17, anti-pT78S79S82, and anti-HA antibodies. Band intensity ratios (pS14S17/HA and pT78S79S82/HA) are shown. Molecular weights are indicated. Data represent two independent experiments.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Construct

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: (A) Schematic for the proximity biotinylation assay to assess the interaction between Vangl2 and Prickle3 ( Pk3). Vangl2 is biotinylated (asterisks) when in proximity to the Pk3 fused to the large N-terminal fragment of a bacterial biotin ligase (BLN). (B) Animal blastomeres of four-to-eight-cell stage embryos were co-injected with 100 pg of FLAG-BLN-Pk3 RNA and 40 pg of HA-Vangl2, HA-Vangl2 Y7,10,12>F (Y>F), or HA-Vangl2 Y7,10,12>E (Y>E) RNAs. At stages 8-9, 20 nl of biotin (0.8 mM) was injected into the blastocoel. Embryos were collected at stage 13, and Vangl2 constructs were pulled down using anti-HA antibody. Biotinylation of Vangl2 and protein expression levels were assessed in independent duplicate samples using anti-biotin, anti-HA, and anti-FLAG antibodies. Data represent three independent experiments.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Cell Surface Biotinylation Assay, Injection, Construct, Expressing

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: (A, B) Vangl2-PTK7 binding is enhanced by non-phosphorylated Vangl2 and reduced by phosphorylated Vangl2. Four-to-eight-cell Xenopus embryos were injected with RNAs encoding HA-Vangl2, HA-Vangl2 Y7Y10Y12>F (Y>F) or HA-Vangl2 Y7Y10Y12>E (Y>E) (40 pg each) as indicated, with or without PTK7-GFP RNA, 100 pg, into four animal blastomeres. Embryos were collected at stage 12 and PTK7-GFP (A) or HA-Vangl2 (B) were immunoprecipitated using GFP-trap or anti-HA antibody, respectively, for Vangl2 analysis (A) or PTK7 analysis (B). Protein levels in pull-downs and lysates were analyzed with anti-GFP and anti-HA antibodies. Irrelevant part of the membrane was removed (A). Data are representative of three experiments.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Binding Assay, Injection, Immunoprecipitation, Membrane

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Neural folding defects were assessed in embryos injected with RNAs encoding PTK7 and different forms of Vangl2 (200 pg each) into two dorsal animal blastomeres (inset in A, red arrows). Dorsal views of representative embryos exhibit no (A), mild (B) or severe (C) disruption of neural tube closure. Co-injection of PTK7 and Y>F Vangl2 RNAs causes stronger neural tube defects (NTDs) compared to the co-injection of the wild type or Y>E Vangl2 RNAs. The degree of neural tube closure was scored at stage 17 by the distance between the opposing neural folds near the brain-spinal cord border (arrowheads), anterior is to the top. Dashed line marks the midline. (D) Quantification showing frequencies of normal neural folds (A), as compared to mild (B) and severe (C) defects. The number of scored embryos is shown in the graph. The significance was determined by full 2×3 Fisher’s exact test that was calculated in Prism 10 software; **** p<0.0001, * p<0.05. Data represent two independent experiments. (E) Model. Graded FGF/FGFR1 activity triggers Vangl2 tyrosine phosphorylation at posterior cell edges, inhibiting the formation of Vangl2-Pk3 and Vangl2-PTK7 complexes, leading to reduced posterior accumulation of Vangl2. PTK7 is proposed to act as a feedback regulator and FGFR1 antagonist, promoting Vangl2 dephosphorylation through phosphatase recruitment.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Disruption, Software, Activity Assay, Phospho-proteomics, De-Phosphorylation Assay

Journal: Journal of hepatology

Article Title: Role of the IgG4-related cholangitis autoantigen annexin A11 in cholangiocyte protection.

doi: 10.1016/j.jhep.2021.10.009

Figure Lengend Snippet: Fig. 1. Annexin A11 is expressed in the cytoplasm and the apical and basolateral plasma membrane of human cholangiocytes. (A) Immunohistochemistry staining for annexin A11 in human liver tissue. (B) Cell surface biotinylation assay followed by immunoblotting for annexin A11 (observed molecular weight: 56 kDa) in sham and ANXA11 KD H69 cholangiocytes. Na+/K+ ATPase is used as loading control, GAPDH serves as proof for adequate separation of biotinylated plasma membrane proteins (no GAPDH) from intracellular proteins in the cell lysate. (C) Quantification of annexin A11 protein levels (7 cell samples of n = 3 independent experiments). (D) ANXA11 gene expression level in sham and ANXA11 KD H69 cholangiocytes (9 cell samples of n = 3 independent experiments). (E) Immu- nofluorescence staining for annexin A11 in human liver tissue. < basolateral cholangiocyte cell membrane, # bile duct lumen. Data are presented as median with interquartile range. Levels of significance: ***p <0.001, ****p <0.0001 (Mann-Whitney U test (C), unpaired t test (D)). KD, knockdown; NB, non-biotin control of sham H69 cholangiocytes. (This figure appears in color on the web.)

Article Snippet: After blocking for 1 hour with 5% normal goat serum in 1% BSA/ TBST (tris-buffered saline with 0.05% (w/v) Tween 20), sections were triple-incubated with anti-human annexin A11 primary antibody (Santa Cruz, Dallas, TX), anti-human ANO1 primary antibody (Abcam, Cambridge, United Kingdom) and anti-human AE2 primary antibody (Santa Cruz, Dallas, TX) 1:50 in 1% BSA/ TBST overnight at 4 C. Sections were then incubated for 1 hour with the following secondary antibodies: anti-rabbit Alexa Fluor 555 (Invitrogen, Waltham, MS), anti-mouse Alexa Fluor 488 (Invitrogen, Waltham, MS) and anti-goat Alexa Fluor 647 (Invitrogen, Waltham, MS) 1:100 in 1% BSA/TBST (at room temperature, protected from light).

Techniques: Clinical Proteomics, Membrane, Immunohistochemistry, Staining, Cell Surface Biotinylation Assay, Western Blot, Molecular Weight, Control, Gene Expression, MANN-WHITNEY, Knockdown

Journal: Journal of hepatology

Article Title: Role of the IgG4-related cholangitis autoantigen annexin A11 in cholangiocyte protection.

doi: 10.1016/j.jhep.2021.10.009

Figure Lengend Snippet: Fig. 3. Annexin A11 is required for ANO1, but not AE2 membrane localization in human cholangiocytes. Cell surface biotinylation assay followed by immunoblotting for (A) ANO1 (observed molecular weight: 75 kDa) or (B) AE2 (observed molecular weight: 150 kDa) in sham and ANXA11 KD H69 chol- angiocytes. Na+/K+ ATPase is used as loading control, GAPDH serves as proof for adequate separation of biotinylated plasma membrane proteins (no GAPDH) from total cell lysate. (C, D) Quantification of protein levels (6 cell samples of n = 3 independent experiments). (E) Baseline intracellular pH of sham and ANO1 KD H69 cholangiocytes (n = 3 independent experiments). (F) Representative full trace of baseline intracellular pH in sham and ANO1 KD H69 cholangiocytes. (G) Immunofluorescence staining for annexin A11, AE2 and ANO1 in human liver. (H) Pearson’s correlation coefficients (7 bile ducts in n = 3 livers). Data are presented as median with interquartile range. Levels of significance: **p <0.01, n.s. not significant (Mann-Whitney U test (C, D), paired t test (E)). KD, knockdown; NB, non- biotin control of sham H69 cholangiocytes. (This figure appears in color on the web.)

Article Snippet: After blocking for 1 hour with 5% normal goat serum in 1% BSA/ TBST (tris-buffered saline with 0.05% (w/v) Tween 20), sections were triple-incubated with anti-human annexin A11 primary antibody (Santa Cruz, Dallas, TX), anti-human ANO1 primary antibody (Abcam, Cambridge, United Kingdom) and anti-human AE2 primary antibody (Santa Cruz, Dallas, TX) 1:50 in 1% BSA/ TBST overnight at 4 C. Sections were then incubated for 1 hour with the following secondary antibodies: anti-rabbit Alexa Fluor 555 (Invitrogen, Waltham, MS), anti-mouse Alexa Fluor 488 (Invitrogen, Waltham, MS) and anti-goat Alexa Fluor 647 (Invitrogen, Waltham, MS) 1:100 in 1% BSA/TBST (at room temperature, protected from light).

Techniques: Membrane, Cell Surface Biotinylation Assay, Western Blot, Molecular Weight, Control, Clinical Proteomics, Staining, MANN-WHITNEY, Knockdown

Journal: Journal of hepatology

Article Title: Role of the IgG4-related cholangitis autoantigen annexin A11 in cholangiocyte protection.

doi: 10.1016/j.jhep.2021.10.009

Figure Lengend Snippet: Fig. 4. Colocalization of annexin A11 and ANO1 is stimulated by elevated intracellular free Ca2+ levels in H69 cholangiocytes. (A) Live-cell imaging of ionomycin treated (10 lM for 15 minutes) annexin A11-mEmerald-over- expressing and ANO1-mCherry-transfected H69 cholangiocytes. (B) Pearson’s correlation coefficient in the region of interest (defined as cells showing annexin A11-mEmerald localization shift and ANO1-mCherry expression) (6 regions of interest of n = 3 independent experiments). Data are presented as median with interquartile range. Level of significance: ****p <0.0001 (unpaired t test). (This figure appears in color on the web.)

Article Snippet: After blocking for 1 hour with 5% normal goat serum in 1% BSA/ TBST (tris-buffered saline with 0.05% (w/v) Tween 20), sections were triple-incubated with anti-human annexin A11 primary antibody (Santa Cruz, Dallas, TX), anti-human ANO1 primary antibody (Abcam, Cambridge, United Kingdom) and anti-human AE2 primary antibody (Santa Cruz, Dallas, TX) 1:50 in 1% BSA/ TBST overnight at 4 C. Sections were then incubated for 1 hour with the following secondary antibodies: anti-rabbit Alexa Fluor 555 (Invitrogen, Waltham, MS), anti-mouse Alexa Fluor 488 (Invitrogen, Waltham, MS) and anti-goat Alexa Fluor 647 (Invitrogen, Waltham, MS) 1:100 in 1% BSA/TBST (at room temperature, protected from light).

Techniques: Live Cell Imaging, Expressing, Transfection

Journal: Journal of hepatology

Article Title: Role of the IgG4-related cholangitis autoantigen annexin A11 in cholangiocyte protection.

doi: 10.1016/j.jhep.2021.10.009

Figure Lengend Snippet: Fig. 5. Serum of a patient with IRC containing anti-annexin A11 IgG1/IgG4-autoantibodies, but not of patients with PSC, inhibits the Ca2+-dependent membrane shift of annexin A11 and reduces plasma membrane localization of ANO1. (A) IgG1/IgG4-autoantibody binding sites on annexin A11 (orange) and Ca2+-binding domains (cyan) as predicted using the ElliPro software tool. (B) Ionomycin-treated (50 lM for 15 minutes) annexin A11-mEmerald-overexpressing H69 cholangiocytes after 3 days of incubation with 20% PSC or IRC patient serum. Proportion of cells showing annexin A11-mEmerald localization shift (7 confocal pictures of n = 3 independent experiments). (C) ANO1-mCherry plasma membrane localization in H69 cholangiocytes after 6 days of incubation with 20% PSC or IRC patient serum. (D) Semiquantitative scoring of ANO1-mCherry plasma membrane localization (1 scorer, n = 3 independent experiments; see also Fig. S5 for scorer 2 and 3). Data are presented as median with interquartile range. Level of significance: **p <0.01, ****p <0.0001 (unpaired t test (B), two-way ANOVA (D)). IRC, IgG4-related cholangitis; PSC, primary sclerosing cholangitis. (This figure appears in color on the web.)

Article Snippet: After blocking for 1 hour with 5% normal goat serum in 1% BSA/ TBST (tris-buffered saline with 0.05% (w/v) Tween 20), sections were triple-incubated with anti-human annexin A11 primary antibody (Santa Cruz, Dallas, TX), anti-human ANO1 primary antibody (Abcam, Cambridge, United Kingdom) and anti-human AE2 primary antibody (Santa Cruz, Dallas, TX) 1:50 in 1% BSA/ TBST overnight at 4 C. Sections were then incubated for 1 hour with the following secondary antibodies: anti-rabbit Alexa Fluor 555 (Invitrogen, Waltham, MS), anti-mouse Alexa Fluor 488 (Invitrogen, Waltham, MS) and anti-goat Alexa Fluor 647 (Invitrogen, Waltham, MS) 1:100 in 1% BSA/TBST (at room temperature, protected from light).

Techniques: Membrane, Clinical Proteomics, Binding Assay, Software, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Subcellular detection of NHE6 variants in recycling endosomes in transfected AP-1 cells. A, AP-1 cells were transiently transfected with mCherry fluorescent protein-tagged NHE6 (NHE6ChFP) WT or CS-linked variants. Forty eight hours post-transfection, cells were incubated with the recycling endosomal marker Alexa Fluor 488–conjugated transferrin (Tf-AF488, 10 μg/ml) for 45 min, fixed in 4% paraformaldehyde, mounted onto glass slides, and examined by confocal microscopy. Images show each channel individually, with merged images of the NHE6ChFP and Tf-AF488 channels. Scale bars represent 10 μm. B, quantitation of the degree of NHE6 overlapping with Tf-AF488 as determined by calculating the thresholded Mander's coefficient (M1) using ImageJ software and the JACoP plugin. Data are plotted as a box chart, with the central white square indicating the mean, the box representing the S.E., and the error bars showing the S.D. (n = 6–8 cells). Significance from WT was determined by one-way repeated measures ANOVA (F value = 6479.8, p value = 5.6 × 10−9), with a post hoc Dunnett's test, ★★★, p < 0.001.

Article Snippet: Next, the cells were incubated with FITC-conjugated human transferrin (Tf–FITC) (Molecular Probes) at 6.6 μg/ml in serum-free α-MEM for 1 h at 37 °C and 5% CO 2 .

Techniques: Transfection, Incubation, Marker, Confocal Microscopy, Quantitation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the functional properties of NHE6 variants. A, biochemical determination of plasma membrane trafficking of NHE6GFP WT or CS-linked variants using a cell-surface biotinylation assay. Cell-surface proteins were labeled with N-hydroxysulfosuccinimidyl–SS–biotin in AP-1 cells expressing the NHE6GFP constructs after 48 h. Total-cell lysates (left panel; protein loading ranged from 10 to 50 μg of protein per sample as indicated below the blot) and biotinylated fractions (right panel; representing 20–100% of plasma membrane proteins extracted per sample) were examined by Western blotting with polyclonal anti-GFP and monoclonal anti-GAPDH antibodies. Representative blots from three experiments are shown. B and C, surface expression and endocytosis of external triple flag tag–labeled NHE6 (3FNHE6HA) constructs in transiently transfected (48 h) AP-1 cells using a cell-based ELISA. Mean intensity fluorescence (M.I.F.) units were determined as a function of the cellular protein concentration and then normalized as percentage (M.I.F. units for WT (100%): 25,100 ± 6,348, n = 4). The surface expression of each construct at time 0 min (before the start of internalization) is charted in B (n = 3–4 experiments). Significance from WT-expressing cells was determined using a one-way repeated measures ANOVA (F value = 463.3, p value = 0.0022), with a post hoc Dunnett's test; *, p < 0.05. Percentage internalization of NHE6 constructs normalized to the zero time point are presented in C and represent the mean ± S.D. (n = 3–4 experiments). The NHE6 variants clustered into two groups: 1) WT, A9S, and R568Q, and 2) L188P, G383D, E547*, and W570*, with variants within each cluster yielding similar statistical values. Significance from WT cells at the 5- and 15-min time points was determined using a one-way ANOVA (F value = 9.43, p value = 4.48 × 10−5), with a post hoc Tukey test; ★, p < 0.05. D, transferrin uptake in HeLa cells transiently transfected (48 h) with GFP or NHE6GFP constructs. The initial uptake (5 min) of Alexa 633–conjugated transferrin (Tf-AF633) was measured in 1 × 104 GFP-positive HeLa cells per experiment by flow cytometry (M.I.F. units for GFP control: 10,204 ± 1554, n = 4). Data were normalized as a percentage and displayed as percent change from GFP control cells. Significance from control cells was determined using a one-way repeated measures ANOVA (F value = 320.7, p value = 3.8 × 10−4), with a post hoc Dunnett's test; ★★, p < 0.001. E, recycling endosomal pH (pHe) was measured in AP-1 cells in the absence or presence of transiently transfected (48 h) NHE6ChFP constructs by fluorescence ratio image analysis of the internalized pH-sensitive probe FITC-conjugated human transferrin (Tf–FITC). Data represent the average endosomal pHe per cell pooled from three separate experiments (8–12 cells per construct/experiment; n = 24–36). Significance was determined by one-way ANOVA (F value = 40.02, p value = 0), with a post-hoc Tukey test; ★★, p < 0.001. Data in B, D, and E are plotted as box charts, with the central white square indicating the mean; the box representing the S.E.; and the error bars showing the S.D. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Next, the cells were incubated with FITC-conjugated human transferrin (Tf–FITC) (Molecular Probes) at 6.6 μg/ml in serum-free α-MEM for 1 h at 37 °C and 5% CO 2 .

Techniques: Functional Assay, Clinical Proteomics, Membrane, Cell Surface Biotinylation Assay, Labeling, Expressing, Construct, Western Blot, FLAG-tag, Transfection, In-Cell ELISA, Fluorescence, Protein Concentration, Flow Cytometry, Control

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: ( A ) An outline of the screening strategy for negative components of Wnt/β-catenin signaling using a HEK293T-TOP-EGFP reporter line. ( B ) Representative images of the reporter cells with or without infection of the GeCKO library. GFP + cells were enriched by FACS. Scale bar, 200 µm. ( C ) Scatter plots showing candidate genes corresponding to sgRNAs enriched. Results of two independent screens are shown. Each dot represents a candidate gene. Y-axis: number of unique sgRNAs for each gene (six sgRNAs per gene in the GeCKO library). X-axis: sgRNA NGS (next generation sequencing) reads for each gene. ( D ) Schematic diagram of TMEM79 protein (predicted). Y280*, a nonsense mutation at tyrosine 280 coded by the matted allele in mice. Figure 1—source data 1. Raw data for .

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Infection, Next-Generation Sequencing, Mutagenesis

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: ( A ) ClustalW alignment of TMEM79/Tmem79 proteins from human ( Homo sapiens ), chimpanzee ( Pan troglodytes ), rat ( Rattus norvegicus ), mouse ( Mus musculus ), chicken ( Gallus gallus ), frog ( Xenopus laevis ), zebra fish ( Danio rerio ), coelacanth ( Latimeria chalumnae ), hemicordate ( Saccoglossus kowalevskii ), great scallop ( Pecten maximus ), leech ( Helobdella robusta ), cauliflower coral ( Pocillopora damicornis ), and sea anemone ( Nematostella vectensis ). Black and grey represent identical and conservative amino acid residues, respectively. Note that the amino terminus before the transmembrane domain 1 (TM1) is poorly conserved and shown only partially. The predicted Tmem79 of Nematostella vectensis appears to lack the 5th transmembrane domain (TM5) that is present in other species listed. Accession numbers for the Tmem79 sequences in the alignment are listed in Materials and methods section. ( B ) The phylogenetic tree showing homology among Tmem79 proteins from species presented in A based on Neighbor-Joining phylogenetic methods. Distances in the cladogram are derived from amino acid substitutions. ( C ) Orthologous comparisons of identity of Tmem79 proteins among species listed in A using ClustalW alignment. ( D ) ClustalW alignment of vertebrate TMEM79/Tmem79 proteins from human, chimpanzee, rat, mouse, chicken, frog, zebra fish, and coelacanth with mouse MAPEG family members Mgst1, Mgst2, Mgst3, Flap and Ltc4s ( ; ). Asterisk indicates two conserved residues within the region of limited sequence similarity corresponding to mouse Tmem79 R332 and Y339.

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Derivative Assay, Sequencing

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: ( A ) siRNA depletion of TMEM79 enhances top-flash reading with or without Wnt3a. A ZNRF3 siRNA serves as a positive control. CM, conditioned medium. TOP-Flash is quantified using RLA (relative luciferase activity), with the control set as 1. ( B ) TMEM79 dose-dependently suppresses top-flash. FOP, a negative control luciferase reporter. EV, empty vector. Inset: immunoblot of TMEM79 proteins, with β-actin as loading control. ( C ) In animal pole explants of Xenopus embryos, TMEM79 inhibits expression of xnr3 induced by Wnt8 but not by β-catenin. TMEM79 does not inhibit expression of target genes (Xbra, vent2, and Gli1) induced by Nodal, FGF, BMP, or Shh, respectively. Ef1α: RT-PCR/loading control; WE, whole embryo; Uninj, uninjected embryo; -RT: no reverse transcriptase. In this and all other figures, gradient bars symbolize different doses of DNA in transfection or mRNA in embryo injection; error bars represent SEM from at least three independent experiments; *p<0.05, **p<0.01, and ***p<0.001, ****p<0.0001 are based on student’s t-tests. Figure 2—source data 1. Raw data for .

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Positive Control, Luciferase, Activity Assay, Control, Negative Control, Plasmid Preparation, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Transfection, Injection

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: ( A and B ) TMEM79 affects neither the cAMP/PKA pathway, assayed by a CRE (cAMP responsive element)-driven reporter, or the EGF/EGFR pathway, assayed by an SRE (serum response element)-(driven reporter). ( C and D ) Depletion of TMEM79 by siRNAs affects neither the CRE- or SRE-driven reporter. ( E ) TMEM79 inhibits the TOP-Flash reporter induced by Wnt3a but not that induced by a GSK3 inhibitor (GSK3i). ( F ) Relative mRNA levels of TMEM79 in HEK293 cells transfected with indicated siRNA. Figure 2—figure supplement 1—source data 1. Raw data for .

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Transfection

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: ( A ) Porcupine inhibitor IWP-2 abolishes Top-Flash activities induced by a TMEM79 siRNA. ( B ) Levels of phospho-LRP6 and phospho-DVL2 are elevated in T79KO and ZRKO cells, and are reduced/reversed by IWP-2, as detected by a phospho-LRP6 (S1490) antibody and the upper DVL2 protein band, respectively. ( C ) Endogenous FZD5 levels are elevated in T79KO and ZRKO cells comparably. FZD null cells (knockout of FZD1-10) serve as control for immunoblotting specificity. ( D ) TMEM79 and ZNRF3 inhibit Wnt signaling independent of each other. ( E ) siRNA depletion of ZNRF3 or TMEM79 further elevates TOP-Flash activities in T79KO or ZRKO cells. ( F ) Co-IP of TMEM79 (Flag-tagged) with endogenous FZD5 but not LRP6. ( G ) TMEM79 is co-IPed with each FZD (FZD1 to 8) but not SMO, and with FZD9 and FZD10 but not SMO . ( H ) TMEM79 reduces the surface level of FZD5 as indicated by cell surface protein biotinylation assay. Surface proteins were labeled by membrane-impermeable biotin and isolated by streptavidin agarose (strep). Either TMEM79 or ZNRF3 reduces the upper band of FZD5 (the matured and biotinylated form) in input and streptavidin-precipitates, suggesting that TMEM79 or ZNRF3 diminishes surface level of FZD5. Note surface-biotinylation of ZNRF3 but not TMEM79, indicating that TMEM79 is not at the plasma membrane. ( I ) TMEM79 co-IPs the immature form of FZD5. ( J ) FZD5-K0 is resistant to down-regulation by TMEM79 or ZNRF3. Figure 3—source data 1. Raw data for .

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Knock-Out, Control, Western Blot, Co-Immunoprecipitation Assay, Cell Surface Biotinylation Assay, Labeling, Membrane, Isolation, Clinical Proteomics

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: ( A ) Depletion of TMEM79 or ZNRF3 via an siRNA in HEK293T cells induces cytosolic β-catenin accumulation, which is prevented by IWP-2. ( B ) Genomic DNA sequencing showing indel (frame shift) mutations of TMEM79 alleles in the T79KO clone picked/used. Two clones were picked and tested with indistinguishable results (data not shown). ( C ) Top-flash reporter is activated comparably and strongly in T79KO cells and ZRKO cells. ( D ) quantification. Phospho-LPR6 (pS1490) and phospho-DVL2 (the upper band) levels are measured and normalized to total LRP6 and DVL2 levels, respectively. ( E ) Overexpression of TMEM79 and ZNRF3 proteins in ZRKO and T79KO cells, respectively. ( F ) TMEM79 is co-IPed with FZD5 (positive control), FZD9, and FZD10, but not SMO (negative control). ( G ) Mature FZD5 (the upper band) is de-glycosylated by PNGase and converted to the lower band as un-glycosylated immature FZD5. ( H ) TMEM79 co-IPs preferentially the lower band of FZD9, likely the immature form, and reduces the level of upper bands of FZD9, which appear to be heterogenous, possibly due to heterogeneity of glycosylation. This is consistent with FZD9 maturation being inhibited by TMEM79. Other FZDs when overexpressed do not exhibit distinct bands of separable electrophoretic mobilities (data not shown) as do FZD5 most overtly and FZD9 to a lesser degree. ( I ) ZNRF3ΔRING, a mutant with the RING domain deletion, co-IPs mature FZD5 and immature FZD5 (possibly during ZNRF3ΔRING’s transit through the ER to PM). Because the full-length ZNRF3 reduces FZD levels, we employed ZNRF3ΔRING, which does not have the E3 ligase function, to avoid fluctuations of the FZD5 level in the co-IP assay. ( J ) Lysosome inhibitor bafilomycin A1 (BAF A1), but not proteasome inhibitor MG132, inhibits TMEM79-induced degradation of immature FZD5. BAF A1 increases both mature and immature forms of FZD5 (compare lanes 1 and 3). BAF A1 blocks TMEM79-induced degradation of immature FZD5, but does not increase mature FZD5 (compare lanes 3 and 4), consistent with the notion that TMEM79 prevents FZD maturation to the PM by shuttling FZD5 to the lysosome. MG132 does not affect levels of immature and mature FZD5 in the absence (compare lanes 1 and 5) or presence of TMEM79 (compare lanes 2 and 6). Thus it appears that ZNRF3 and RNF43 promote degradation of mature FZD5 through the lysosome , whereas TMEM79 promotes degradation of immature FZD5 through the lysosome. Figure 3—figure supplement 1—source data 1. Raw data for .

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: DNA Sequencing, Clone Assay, Over Expression, Positive Control, Negative Control, Glycoproteomics, Mutagenesis, Co-Immunoprecipitation Assay

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: ( A to D ) Immunofluorescence microscopy of endogenous TMEM79 (red) with ER, PM, lysosome, and Golgi markers (green), respectively, with the nucleus labeled by DAPI (blue). The z-axis is shown on right and underneath. The white scale bar represents 10 µm. Fluorescence intensity of TMEM79 and the respective marker is measured between the two arrowheads in the lower left panel and plotted at lower right. ( A ) TMEM79 is localized at the ER. TMEM79 and calreticulin (an ER marker) overlap extensively in images and intensity traces. ( B ) TMEM79 is not present at the PM, which is visualized by β-catenin-EGFP (produced from the endogenous β-catenin gene with an in-frame knock-in of the EGFP gene, see Materials and methods). Note that β-catenin is predominantly localized at the PM in epithelial cells. ( C ) Some TMEM79 is observed at the lysosome. TMEM79 and LAMP1 (lysosomal-associated membrane protein 1) overlap occasionally. ( D ) TMEM79 is hardly observed at the Trans Golgi and exhibits little or minor overlap with TGN46 (trans-Golgi marker 46).

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Immunofluorescence, Microscopy, Labeling, Fluorescence, Marker, Produced, Knock-In, Membrane

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: ( A ) TMEM79 is not detectable by the TMEM79 antibody without cell permeabilization. Note that the antibody (Key Resources Table) recognizes the TMEM79 C-terminus , which is either in the ER lumen, or extracellular if TMEM79 were to be on the PM. This result is consistent with TMEM79 being an ER- but not PM-resident protein. ( B ) T79KO cells lack TMEM79 staining completely, demonstrating specificity of the TMEM79 antibody for immunostaining. Scale bar = 10 μm.

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Staining, Immunostaining

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: ( A ) Tandem affinity purified proteins were resolved by SDS-PAGE and visualized by silver staining. The same tandem IP procedure of EGFP-expressing cell extracts serves as a negative control. The TMEM79 bait protein is marked, as is the gel fraction that corresponds to the molecular weight of USP8. ( B ) List of candidate proteins from tandem affinity purification-coupled mass spectrometry. Note that only a few visible fractions/bands in A were excised and pooled together for mass spectrometry analyses (see Materials and methods). The list is therefore not intended to be a comprehensive representation of all candidate TMEM79-associated proteins. ( C ) Genomic DNA sequencing showing indel (frame shift) mutations of USP8 alleles in a USP8 KO clone picked/used. Two clones were picked and tested with indistinguishable results (data not shown). ( D ) ZNRF3 inhibits top-flash reporter activity effectively in USP8 KO cells. ( E ) FZD5 co-IPs USP8 in the absence of TMEM79 in T79KO cells. ( F ) Overexpression of TMEM79 does not affect FZD5 co-IP of USP8. This experiment largely rules out a scenario that TMEM79 inhibits USP8 deubiquitination of FZD5 by competing with FZD5 for USP8. TMEM79 is also co-IPed by FZD5. ( G ) USP8 co-IPs TMEM79 in in FZD null cells. Figure 5—figure supplement 1—source data 1. Raw data for .

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Affinity Purification, SDS Page, Silver Staining, Expressing, Negative Control, Molecular Weight, Mass Spectrometry, DNA Sequencing, Clone Assay, Activity Assay, Over Expression, Co-Immunoprecipitation Assay

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: ( A ) Schematic diagram of USP8. USP8N, USP8 N terminal fragment; USP8C, USP8 C terminal fragment. Known domains including DUB and a 14-3-3-binding motif are depicted. ( B ) Activation of Top-Flash by USP8C is inhibited by IWP-2 or by TMEM79. ( C ) TMEM79 is co-IPed by USP8C or USP8CΔ (with the 14-3-3 binding motif deleted), but not by USP8N, suggesting that USP8C (but not its 14-3-3 binding motif) is involved in TMEM79 interaction. ( D ) Schematic diagram of TMEM79 and its deletion mutants. Five transmembrane (TM) regions are depicted. ( E ) TMEM79 (FL, full length) or TMEM79-ΔC, but none of the other TMEM79 mutants, co-IPs USP8C. ( F ) TMEM79 or TMEM79-ΔC, but none of the other TMEM79 mutants, down-regulates the level of mature FZD5. ( G ) The Wnt response in USP8-KO cells is reduced and is not inhibited by TMEM79. ( H and I ) TMEM79 inhibits USP8C deubiquitination of FZD5 ( H ), but not SMO ( I ). HEK293T cells were cotransfected with V5-FZD5 and His-ubiquitin, and in combination with USP8C, TMEM79 or ZNRF3 as indicated. Ubiquitinated proteins were precipitated by Ni-NTA (nickel-nitrilotriacetic acid–agarose beads for His tag pull-down), followed by wash in 7M urea to fully disassociate all protein complexes. Ubiquitinated V5-FZD5 was then precipitated by anti-V5 agarose beads. These multiple steps ensured detection of ubiquitinated V5-FZD5 only, but not V5-FZD5-associated proteins that are also ubiquitinated. Levels of ubiquitinated FZD5 were increased by TMEM79 or ZNRF3 co-expression whereas USP8C deubiquitinated FZD5. TMEM79 inhibited USP8C deubiquitination of FZD5 and restored FZD5 ubiquitination ( H ). USP8C also deubiquitinated SMO, but TMEM79 had no effect on USP8C deubiquitination of SMO ( I ). ( J ) USP8 or USP8C preferentially co-IPs the immature form of FZD5 (the lower band). Figure 5—source data 1. Raw data for .

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Binding Assay, Activation Assay, Ubiquitin Proteomics, Expressing

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: ( A ) Tmem79 is expressed throughout Xenopus embryogenesis from 1 cell (fertilized egg) to stage (st) 30, spanning blastula, gastrula, neurula and tailbud/organogenesis stages, as assayed by RT-PCR. ( B ) Tmem79 is expressed in dissected animal, vegetal, dorsal, and ventral regions (as schemed in drawing) of early gastrula embryos (stage 10.5). xnr3 is a control for dorsal. ( C ) Tmem79 expression during Xenopus embryogenesis by whole-mount in situ hybridization. An apparent lack of hybridization signals in vegetal pole and derived tissues is likely technical due to yolk. Top row (c1 to c6) Tmem79 is expressed in the animal pole at the four-cell stage and in naive ectoderm at blastula stages: (c1) Dorsal-animal view; (c2 to c4) Lateral-animal views; (c5 and c6) Lateral and bisected views. Second row (c7 to c12) Tmem79 is expressed broadly in naive ectoderm at early gastrula stages: (c7 and c8) dorsal-vegetal and lateral views with the arrow pointing to the dorsal; (c9) bisected view with an enlarged view (c10) showing expression in naive ectoderm; (c11 and c12) lateral and bisected views. Anterior to top in c11. Third row (c13 to c18) Tmem79 shows dynamic expression from late gastrula to neurula stages: (c13 and c14) lateral and bisected views showing broad expression including in neural plate. Anterior to top in c13 and left in c14. (c15 to c17) Dorsal views. Anterior to top. Tmem79 expression is downregulated in neural plate while remains strong in epidermis, as seen also in a cross-section (c18) along the dashed line in c17. (c18) an enlarged cross-section view with dorsal on top. Bottom row (c19 to c23) Tmem79 is expressed during organogenesis: (c19 and c20) Lateral and anterior views showing expression in the cement gland. (c21 and c22) A lateral view with an enlarged view (anterior to right) and (c23) a ventral view (anterior to top) showing expression in primitive kidney (black arrows) and otic vesicle (white arrows). ( D and E ) Dorsal-animal (naive ectoderm) injection of Tmem79MO causes anterior deficiency, which is rescued/over-rescued by co-injection of mouse Tmem79 mRNA or Usp8MO or β-cateninMO. CoMO, Control Morpholino. Anterior to right ( E ). Also see . ( F ) Tmem79MO reduces neural plate formation and reciprocally expands epidermal differentiation, reflected by Sox2 and cytokeratin expression domains at Stage 16, respectively. These changes are rescued by Tmem79 mRNA or Usp8MO. Anterior to right. Also see . ( G ) Neural induction by Noggin in animal pole explants is prevented by Tmem79MO, whose effect is rescued by Tmem79 mRNA or Usp8MO or β-cateninMO. Pan-neural (n-cam), anterior (bf1, otx2, and six3) and mid/hind (en2 and krox20) brain markers and a neuronal differentiation marker (n-tub) are assayed, as is an epidermal marker (keratin).

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing, In Situ Hybridization, Hybridization, Derivative Assay, Injection, Marker

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: ( A ) A Tmem79 sense probe as a negative control does not show any hybridization signal in embryos at different stages. ( B ) Dorsal injection of Tmem79 mRNA induces an enlarged head in >50% embryos and spina bifida in another about 30% embryos. Anterior to right. Also see . ( C ) Tmem79MO inhibits protein synthesis from the Xenopus Tmem79 (xTmem79) mRNA, but not of the mouse Tmem79 (mT79) mRNA, which is used in rescue experiments. GAPDH is a protein loading control. ( D ) Usp8MO inhibits protein synthesis from the Xenopus Usp8 (xUsp8) mRNA, but not from a mutant Usp8 (x-mutUsp8) mRNA, in which several nucleotides in xUsp8 5’-UTR targeted by the MO have been altered. ( E ) Dorsal-animal injection of Tmem79 MO reduces expression of anterior and mid/hind brain markers Bf1, Krox20, and En2, an anterior (cement gland) marker Xag (each seen in the anterior view), and a neuronal marker n-tubulin (see in the dorsal view), examined at stage 16. Co-injection of mTmem79 with Tmem79MO rescues expression of these markers. Also see . ( F ) Dorsal-animal injection of β-cateninMO or Usp8MO leads to enlarged head formation at the tailbud stage (anterior to right), supporting Usp8 acting positively in Wnt/β-catenin signaling in vivo. Also see . ( G ) Dorsal-animal injection of Tmem79MO affects neither expression of head organizer genes (Goosecoid, Chordin, Dkk1, and Lim1) nor that of dorsal genes (xnr3 and xnot) at stages 10.5–11. Vegetal views with dorsal to top. Also see . ( H ) Dorsal-animal injection of Tmem79MO reduces expression of neural crest markers FoxD3 and snail1 examined at stage 16. Also see .

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Negative Control, Hybridization, Injection, Control, Mutagenesis, Expressing, Marker, In Vivo

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: ( A to D ) Dorsal (axial mesoderm) injection of Tmem79MO causes spina bifida scored at stage 26 ( A and B ), and a lack of CE movements of axial mesoderm, visualized by a widened (not elongated) xNot-expressing axial tissue and an open blastopore at stage 12 ( C and D ). Axial mesoderm elongation is rescued by Tmem79 mRNA ( C and D ). Anterior to right. Also see . ( E to G ) Axial mesoderm induced by activin in animal pole explants exhibits CE movements, visualized by explant elongation and quantified by a length/width (L/W) ratio ( E and F ). Control explants are round with a L/W ratio of 1 while explants (axial mesoderm induced by activin) elongate with a L/W ratio of 3 ( E and F ). Explants with Tmem79MO poorly elongate but elongation is rescued by Tmem79 mRNA or Usp8MO ( E and F ). Also see . Note that Tmem79MO alone or together with Usp8MO does not affect activin-induced axial mesoderm gene expression ( G ).

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Injection, Expressing, Control, Gene Expression

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: FZD is synthesized in the ER and is ubiquininated (by an unknown E3 ligase, not depicted). FZD deubiquitination by USP8 permits FZD to traffic to Golgi and PM ultimately. ER-resident TMEM79 acts by complexing with FZD and USP8 and inhibiting USP8 deubiquitination of FZD, thereby ensuring ubiquitinated FZD in the ER to traffic to the lysosome for degradation. For FZD that escapes TMEM79-mediated degradation and matures to the PM, ZNRF3 promotes FZD ubiquitination at the PM, leading to endosomal-lysosomal degradation of FZD. These two complementary mechanisms ensure a rate-limiting amount of FZD at the PM.

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Synthesized, Ubiquitin Proteomics

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: Tmem79 and Usp8 are present throughout deuterostomes while exhibit frequent and similar losses in protostomes. Note that both genes are found in cnidaria and thus was likely present in the common cnidarian and bilaterian ancestor . Preservation of Tmem79 in protostomes is always accompanied by preservation of Usp8 , but not vice versa. One such example is in arthropods: Tmem79 is absent but Usp8 is present in Drosophila while the Acari has Tmem79 and Usp8 . Neither gene is found and was probably lost in Xenacoelomorpha.

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Preserving

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet:

Article Snippet: Gene ( M. musculus ) , Tmem79 , Origene , MR206106 , .

Techniques: Luciferase, Knock-In, Knock-Out, Transfection, Construct, Transduction, FLAG-tag, Recombinant, Plasmid Preparation, Sequencing, Negative Control, Injection, Reporter Assay, Silver Staining, Reverse Transcription, Labeling, PCR Cloning, Software, Agarose Gel Electrophoresis

Journal: Journal of Biological Chemistry

Article Title: The Chemokine Receptor CCR1 Is Constitutively Active, Which Leads to G Protein-independent, β-Arrestin-mediated Internalization

doi: 10.1074/jbc.m113.503797

Figure Lengend Snippet: FIGURE1.CCR1expressionissufficienttoinducebasalmigrationandinhibitcAMPformation.MurineL1.2cellsstablytransfectedwitheitherCCR1,CCR2, CCR5, or CCR10 (A) or human THP-1 cells endogenously expressing CCR1 (B) were placed in a microchemotaxis chamber, and the number of cells that spontaneously migrated into the lower chamber after 2 h at 37 °C was measured. The effect of the CCR1-specific inhibitor BX-471 and PT was also determined. The percentage of cells that migrated was calculated as the ratio of cells in the lower chamber in the microchemotaxis well to the number of cells initially added to the upper chamber. Data are the mean S.D. (error bars). C, basal F-actin content of L1.2 cells stably transfected with chemokine receptors or left untransfected (u.t.). Cells were permeabilized and stained with Alexa Fluor 488-phalloidin; results of a representative experiment performed in triplicate are plotted as -fold change over untransfected control. D, luminescence of pGloSensorTM-22F/HEK293 cells transiently transfected with 1.0 g of CCR1 (Œ), M3 (U), or empty pcDNA3.1 () vector. The signal from a representative experiment was measured in triplicate following incubation of cells with 4% GloSensorTM cAMP reagent for 1 h at 37 °C and stimulation with 10 M forskolin. E, gene dosage experiment performed in triplicate in which increasing amounts of HA-CCR1 orHA-M3inpcDNA3.1vectorsweretransfectedintopGloSensorTM-22F/HEK293cellsandassayedasinC.DatawereplottedasthemeanS.D.usingGraphPad Prism (GraphPad Software), and the statistical significance was calculated using an unpaired Student’s t test or one-way analysis of variance with Dunnett’s post-test: **, p 0.01; ***, p 0.0001.

Article Snippet: Cell surface receptor was labeled with mouse anti-CCR1 antibody (clone 53504, R&D Systems) for 30min on ice inwash buffer (DMEM, 0.5%BSA, 10 mM HEPES, pH 7.4), unbound antibody was washed away with cold wash buffer, and then the medium was replaced with prewarmed wash buffer for specified periods of time.

Techniques: Expressing, Stable Transfection, Transfection, Staining, Control, Plasmid Preparation, Incubation, Software

Journal: Journal of Biological Chemistry

Article Title: The Chemokine Receptor CCR1 Is Constitutively Active, Which Leads to G Protein-independent, β-Arrestin-mediated Internalization

doi: 10.1074/jbc.m113.503797

Figure Lengend Snippet: FIGURE 2. Expression of chemokine receptors in various cell lines. A, expression of CCR1, CCR2, CCR5, and CCR10 in stably transfected L1.2 cells. Cell surface receptor expression was determined using receptor-specific antibodies conjugated to PE and analyzed via flow cytometry. Tinted areas demonstrate receptor expression, and unfilled areas demonstrate isotype control antibody binding. B, expression of HA-CCR1 and HA-M3 transiently transfected into HEK293 cells stably expressing the pGloSensor-22F construct. Cell surface receptor levels were detecting using an antibody directed against the HA epitope tag conjugated to PE and analyzed via flow cytometry. Data shown from a representative experiment in triplicate as median fluorescence intensity (MFI). C, mean relative fluorescence values S.D. (error bars) of HEK293t cells transiently transfected in triplicate with CCR1-YFP, CCR2-YFP, CCR5-YFP, or 2AR-YFP in the basal -arrestin-2 BRET association assay. D, effect of chemokine agonist on the BRET1net signal over time between -arrestin-2-Rluc and either CCR1-YFP (●), CCR2-YFP (f), or CCR5-YFP (Œ). CCR1- and CCR5-expressing HEK293t cells were treated with 100 nM CCL14, whereas CCR2-expressing cells were treated with 100 nM CCL7. Data shown are from a representative experiment performed in triplicate.

Article Snippet: Cell surface receptor was labeled with mouse anti-CCR1 antibody (clone 53504, R&D Systems) for 30min on ice inwash buffer (DMEM, 0.5%BSA, 10 mM HEPES, pH 7.4), unbound antibody was washed away with cold wash buffer, and then the medium was replaced with prewarmed wash buffer for specified periods of time.

Techniques: Expressing, Stable Transfection, Transfection, Cell Surface Receptor Assay, Flow Cytometry, Control, Binding Assay, Construct, Fluorescence, Histone Association Assay

Journal: Journal of Biological Chemistry

Article Title: The Chemokine Receptor CCR1 Is Constitutively Active, Which Leads to G Protein-independent, β-Arrestin-mediated Internalization

doi: 10.1074/jbc.m113.503797

Figure Lengend Snippet: FIGURE 4. CCR1 is constitutively phosphorylated. HEK293t cells expressing FLAG-CCR1 were orthotopically labeled with the 32P radioisotope and either leftunstimulatedorstimulatedwith1MCCL14for3or6min(top).Thearrow indicates the band corresponding to the correct molecular weight for CCR1. Receptor levels were measured by Western blot using anti-FLAG-HRP (Sigma) and chemiluminescent detection (bottom). u.t., untransfected.

Article Snippet: Cell surface receptor was labeled with mouse anti-CCR1 antibody (clone 53504, R&D Systems) for 30min on ice inwash buffer (DMEM, 0.5%BSA, 10 mM HEPES, pH 7.4), unbound antibody was washed away with cold wash buffer, and then the medium was replaced with prewarmed wash buffer for specified periods of time.

Techniques: Expressing, Labeling, Molecular Weight, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Chemokine Receptor CCR1 Is Constitutively Active, Which Leads to G Protein-independent, β-Arrestin-mediated Internalization

doi: 10.1074/jbc.m113.503797

Figure Lengend Snippet: FIGURE 5. CCR1 is constitutively associated with -arrestin-2. A, HEK293t cells were transiently transfected with -arrestin-2-Rluc and pcDNA3.1 (white bar) or -arrestin-2-Rluc and CCR1-YFP (black bar), and the BRET1 ratio was measured 48 h later at room temperature in the absence of ligand stimulation. B, comparisonofthebasalBRET1netsignalfromHEK293tcellsco-expressing-arrestin-2-RlucandCCR1-,CCR2-,CCR5-,or2AR-YFP.TheeffectofCCR1inhibition with 1 M BX-471 and blockade of Gi/o signaling with 200 ng/ml PT on the basal BRET1net signal is also shown. C, comparison of the basal BRET1net signal between -arrestin-1-Rluc (white bar) and -arrestin-2-Rluc (black bar) with either CCR1-YFP or CCR5-YFP. D, the basal BRET1net signal between CCR1-YFP and -arrestin-2-Rluc with increasing levels of -arrestin-2-GFP expression. The fold increase in -arrestin-2-GFP levels on the x axis is the ratio of the micrograms of -arrestin-2-GFP vector to -arrestin-2-Rluc vector transfected into cells co-expressing CCR1-YFP. The data above (A–D) are plotted as the mean S.D. (error bars) of a representative experiment done in triplicate. Statistical significance was calculated using an unpaired t test (A) or one-way analysis of variance with Dunnett’s multiple comparisons test (B–D) (*, p 0.05; **, p 0.01). E, HeLa cells were cultured on fibronectin-coated coverslips and transfected with -arrestin-2-GFP alone (far left panel) or with co-transfected with CCR1-mCherry (three right panels). Cells were washed, fixed, and imaged using a confocal microscope. Co-localization is indicated as yellow in the merged image. F, HEK293t cells were transiently transfected with FLAG-CCR1 or pcDNA3.1 (far right lanes) and increasing levels of -arrestin-2-HA and treated with PBS or 100 nM CCL14 for 15 min at 37 °C. CCR1 and any associated proteins were immunopre- cipitated (IP), and the presence of receptor and -arrestin-2-HA was measured by Western blot (IB).

Article Snippet: Cell surface receptor was labeled with mouse anti-CCR1 antibody (clone 53504, R&D Systems) for 30min on ice inwash buffer (DMEM, 0.5%BSA, 10 mM HEPES, pH 7.4), unbound antibody was washed away with cold wash buffer, and then the medium was replaced with prewarmed wash buffer for specified periods of time.

Techniques: Transfection, Expressing, Comparison, Plasmid Preparation, Cell Culture, Microscopy, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Chemokine Receptor CCR1 Is Constitutively Active, Which Leads to G Protein-independent, β-Arrestin-mediated Internalization

doi: 10.1074/jbc.m113.503797

Figure Lengend Snippet: FIGURE 6. Constitutive internalization of CCR1 is mediated by -arrestin-2. Wild-type and -arrestin-2-deficient MEFs were transiently transfected on coverslips with HA-CCR1, and cell surface receptor was prelabeled with anti-HA Alexa Fluor 594. Cells were then warmed with serum-free medium without agonist for 30 min, and the extent of receptor internalization was observed using a confocal microscope.

Article Snippet: Cell surface receptor was labeled with mouse anti-CCR1 antibody (clone 53504, R&D Systems) for 30min on ice inwash buffer (DMEM, 0.5%BSA, 10 mM HEPES, pH 7.4), unbound antibody was washed away with cold wash buffer, and then the medium was replaced with prewarmed wash buffer for specified periods of time.

Techniques: Transfection, Cell Surface Receptor Assay, Microscopy

Journal: Journal of Biological Chemistry

Article Title: The Chemokine Receptor CCR1 Is Constitutively Active, Which Leads to G Protein-independent, β-Arrestin-mediated Internalization

doi: 10.1074/jbc.m113.503797

Figure Lengend Snippet: FIGURE 7. CCR1 is constitutively associated with Gi and forms a basal complex with G protein and -arrestin. A, HEK293 cells were transiently co-transfected with CCR1-YFP or M3-YFP and Gi-Rluc with insertion of the luciferase at position 60 (Gi-60-Rluc, black bar) or 122 (Gi-122-Rluc, white bar) to test for effects of Rluc orientation. Forty-eight hours post-transfection, the basal BRET1net value was measured. B, BRET saturation assay in which the expression level of Gi-60-Rluc (f), Gi-122-Rluc (Œ), or G12/13-Rluc (●) was kept constant while the expression of CCR1-YFP was continually increased. The respective curves indicate a specific associ- ation between CCR1-YFP and either of the Gi-Rluc constructs but not for G12/13-Rluc. C, FLAG-CCR1/HEK293/TetO cells in which CCR1 expression can be induced withdoxycyclineweretransfectedwithbothGi-122-Rlucand-arrestin-2-YFP.ThechangeintheBRET1netsignalbetweenGiand-arrestin-2intheabsence(white bar) and presence (black bar) of 2 g/ml doxycycline is shown. The statistical significance was calculated using an unpaired t test (**, p 0.01) (GraphPad Prism). D, the same cells as in C were stimulated with 1 M CCL14, and the effect of the agonist on the BRET1net signal over time is shown. Error bars, S.D.

Article Snippet: Cell surface receptor was labeled with mouse anti-CCR1 antibody (clone 53504, R&D Systems) for 30min on ice inwash buffer (DMEM, 0.5%BSA, 10 mM HEPES, pH 7.4), unbound antibody was washed away with cold wash buffer, and then the medium was replaced with prewarmed wash buffer for specified periods of time.

Techniques: Transfection, Luciferase, Saturation Assay, Expressing, Construct

Journal: Journal of Biological Chemistry

Article Title: The Chemokine Receptor CCR1 Is Constitutively Active, Which Leads to G Protein-independent, β-Arrestin-mediated Internalization

doi: 10.1074/jbc.m113.503797

Figure Lengend Snippet: FIGURE8.CCR1formsaspecifichomo-oligomer.A,BRETsaturationassayin which energy donor (CCR1-Rluc) expression levels are kept constant while the expression of energy acceptor (receptor-YFP) is continually increased in order to compare the homo-oligomerization of CCR1 (●) with hetero-oligo- merization with the M3 (Œ) or GABA(B2) (f) receptor. A non-linear and satu- rable curve is indicative of a specific interaction between the two proteins being studied. B, type II BRET saturation assay in which the expression ratio between the energy donor (CCR1-Rluc) and energy acceptor (receptor-YFP) is kept constant while the overall expression of both proteins is continually increased in order to compare homo-oligomerization of CCR1 (●) with hetero-oligomerization with the M3 (Œ) receptor. A linear relationship with a non-zero intercept is indicative of a specific interaction. Error bars, S.D.

Article Snippet: Cell surface receptor was labeled with mouse anti-CCR1 antibody (clone 53504, R&D Systems) for 30min on ice inwash buffer (DMEM, 0.5%BSA, 10 mM HEPES, pH 7.4), unbound antibody was washed away with cold wash buffer, and then the medium was replaced with prewarmed wash buffer for specified periods of time.

Techniques: Expressing, Saturation Assay

Journal: Journal of Biological Chemistry

Article Title: The Chemokine Receptor CCR1 Is Constitutively Active, Which Leads to G Protein-independent, β-Arrestin-mediated Internalization

doi: 10.1074/jbc.m113.503797

Figure Lengend Snippet: FIGURE9.CCR1-mediatedinternalizationofCCL7-Cy3BandeffectofGproteininactivation.CCR1/L1.2cellsweretreatedintheabsenceorpresenceof0.2 g/ml PT or 100 M BX-471 for 1 h prior to incubation with CCL7-Cy3B for up to 30 min at 37 °C. CCR5/L1.2 and untransfected (u.t.)/L1.2 cells were included as controls. Internalization of CCL7-Cy3B was measured in triplicates as the median fluorescence intensity (MFI) of cells analyzed on a guava easyCyteTM flow cytometer. The data are displayed as -fold change of median fluorescence intensity over unstimulated cells. The statistical significance of the difference in -fold change in median fluorescence intensity is displayed for the 30 min time point and compared with untreated CCR1/L1.2 using a two-way analysis of variance with Bonferroni post-tests. **, p 0.01 (GraphPad Prism). Error bars, S.D.

Article Snippet: Cell surface receptor was labeled with mouse anti-CCR1 antibody (clone 53504, R&D Systems) for 30min on ice inwash buffer (DMEM, 0.5%BSA, 10 mM HEPES, pH 7.4), unbound antibody was washed away with cold wash buffer, and then the medium was replaced with prewarmed wash buffer for specified periods of time.

Techniques: Incubation, Fluorescence, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: The Chemokine Receptor CCR1 Is Constitutively Active, Which Leads to G Protein-independent, β-Arrestin-mediated Internalization

doi: 10.1074/jbc.m113.503797

Figure Lengend Snippet: FIGURE10.ModelofCCR1constitutiveactivity.CCR1expressionissufficientforinducingbasalmigrationandGproteinsignaling,whichcanbeblockedwith a CCR1-specific inhibitor or PT treatment (left). At the same time, CCR1 is also constitutively phosphorylated, leading to -arrestin-2 recruitment, receptor internalization, and recycling (middle). The fate of the internalized receptor and whether it is sent for degradation or eventually recycled back to the cell surface in the presence of ligand stimulation remains to be determined. CCR1 inhibition with BX-471 was unable to block constitutive internalization or prevent basal association with -arrestin-2. Additionally, a preformed complex that brings CCR1, Gi, and -arrestin-2 into close proximity may provide precise regulation of signal transduction by a constitutively active or agonist-activated receptor. The observation that CCR1 forms a homo-oligomer may also explain how the receptor is physically able to form concurrent interactions with these intracellular proteins (right). AC, adenylyl cyclase.

Article Snippet: Cell surface receptor was labeled with mouse anti-CCR1 antibody (clone 53504, R&D Systems) for 30min on ice inwash buffer (DMEM, 0.5%BSA, 10 mM HEPES, pH 7.4), unbound antibody was washed away with cold wash buffer, and then the medium was replaced with prewarmed wash buffer for specified periods of time.

Techniques: Inhibition, Blocking Assay, Transduction

Journal: Toxins

Article Title: Interaction of Clostridium perfringens Epsilon Toxin with the Plasma Membrane: The Role of Amino Acids Y42, Y43 and H162

doi: 10.3390/toxins14110757

Figure Lengend Snippet: Western blot analysis of SDS-resistant Etx oligomers in hRBCs. ( a ) Human RBCs were incubated with trypsin-activated Etx variants or buffer only at room temperature or 37 °C for 1 h. Following detergent solubilisation, toxin complexes (30 µg per lane) were separated by SDS-PAGE and oligomerization was assessed by immunoblotting with anti-Etx polyclonal antibody. Lanes 1 and 7: Molecular weight marker; Lanes 2–6: hRBCs incubated with trypsin-activated Etx-wild type, Etx-H162A, Etx-Y42A, Etx-Y43A or buffer only, respectively at room temperature; Lanes 8–12: hRBCs incubated with trypsin-activated Etx-wild type, Etx-H162A, Etx-Y42A, Etx-Y43A or buffer only, respectively at 37 °C. Arrows indicate Etx monomer (m), Etx oligomer (o) and loading control (lc). FLOT2 antibody was used as loading control. One representative immunoblot out of three is shown for each experiment. The molecular masses (kDa) of protein standards are shown to the left of the blot. ( b ) Normalised, mean oligomer yields for Etx-H162A, Etx-Y42A and Etx-Y43A were determined by calculating the % oligomerisation relative to cells treated with wild type Etx (100%) using Image Studio ver. 5.2 software. Values represent the means of triplicate experiments.

Article Snippet: For loading control, the mouse monoclonal Flotillin 2 (FLOT2) antibody (Proteintech, Rosemont, IL, USA; catalogue number 66881-11g, lot 10008101, Genbank Accession no. BC017292) and the IRDye ® 680RD goat (polyclonal) anti-mouse IgG (H + L) secondary antibody (LI-COR Biosciences, Lincoln, NE, USA; catalogue number 925-68070) were used at 1:2000 and 1:5000 dilutions, respectively.

Techniques: Western Blot, Incubation, SDS Page, Molecular Weight, Marker, Control, Software

Journal: Pathogens

Article Title: Acanthamoeba castellanii : Non-Steroidal Anti-Inflammatory Drugs Affect Adhesion, Motility, and Encystment, Suggesting a Link with a gp63-like Protein Candidate

doi: 10.3390/pathogens15030263

Figure Lengend Snippet: Expression of gp63 and organization of the actin cytoskeleton in Acanthamoeba castellanii incubated with discontinuous micropatterns of mouse brain tissue (BTDM). ( A ) Western blot analysis of gp63-like protein (Leishmanolysin) expression in trophozoites. Lane 1 corresponds to membrane fractions from trophozoites incubated without BTDM, and lane 2 corresponds to trophozoites incubated with BTDM for 1 h. gp63 was detected using a commercial anti-gp63 antibody. ( B ) Confocal fluorescence microscopy images showing F-actin (rhodamine–phalloidin, red) and gp63-like protein (green). Co-localization appears in yellow. ( a – e ). Trophozoites cultured without BTDM ( a , c ) and trophozoites cultured with BTDM for 1 h ( b , e ). Higher magnification of individual cells highlighting co-localization (( c ) = without BTDM; ( e ) = with BTDM). In ( d , f ), corresponds to DIC analysis. Arrows indicate representative areas of co-localization. Scale bars = 20 μm. ( C ) Quantitative analysis of the colocalization between filamentous actin and the gp63-like protein, based on fluorescence signal intensity, in trophozoites cultured with or without BTDM stimulation. Non-stimulated trophozoites exhibited a mean F-actin/gp63-like protein colocalization of approximately 3.8, whereas BTDM-stimulated trophozoites showed an increase to approximately 20% ( n = 6 trophozoites). Total fluorescence intensity and colocalization between the red (F-actin) and green (gp63-like protein) signals were quantified using ImageJ 2 software. Data represent three independent biological replicates ( n = 3) and are expressed as mean ± SEM.

Article Snippet: Samples were incubated with a commercial monoclonal anti- Leishmania major gp63 antibody (anti- Leishmania major monoclonal antibody (Cat. No. CLP005A, (CEDARLANE Laboratories Limited, Burlington, ON, Canada;), which recognizes conserved epitopes within the gp63-family metalloproteases in their native state, followed by appropriate fluorescent secondary antibodies.

Techniques: Expressing, Incubation, Western Blot, Membrane, Fluorescence, Microscopy, Cell Culture, Software

Journal: Pathogens

Article Title: Acanthamoeba castellanii : Non-Steroidal Anti-Inflammatory Drugs Affect Adhesion, Motility, and Encystment, Suggesting a Link with a gp63-like Protein Candidate

doi: 10.3390/pathogens15030263

Figure Lengend Snippet: Confocal immunofluorescence microscopy analysis of gp63-like protein and actin distribution in trophozoites and cysts. ( A ) Representative confocal images showing actin staining (orange) and gp63-like protein labeling (green) in trophozoites and cysts. Actin displayed a more intense signal in trophozoites, whereas gp63 was detected in both stages. ( B ) Merged images illustrating partial colocalization of gp63 and actin, predominantly at the trophozoite plasmatic membrane. In the case of ectocyst, the presence of gp63-like protein is abundant (arrow). Differential interference contrast (DIC) images confirm cyst and trophozoite morphology. ( C ) Confocal images of mature cysts showing gp63-like protein localization in both the ectocyst and endocyst layers (arrows). Actin signal was more abundant in the ectocyst, as observed in merged images. DIC images highlight the differentiation of cyst layers. ( D ) Calcofluor staining (blue), analyzed by confocal microscopy, uniformly cyst walls. gp63-like protein labeling (green) was detected in cysts with well-defined ectocyst and endocyst layers, while DIC imaging revealed a subpopulation of calcofluor-positive cysts lacking detectable gp63-like protein signal. ( E , F ) Confocal gp63 localization at the operculum, as evidenced by immunofluorescence and confirmed by DIC imaging (arrows). ( E ) Higher-magnification confocal images show gp63-like protein localization in the ectocyst, endocyst, and operculum (arrows). Scale bars = 10 μm.

Article Snippet: Samples were incubated with a commercial monoclonal anti- Leishmania major gp63 antibody (anti- Leishmania major monoclonal antibody (Cat. No. CLP005A, (CEDARLANE Laboratories Limited, Burlington, ON, Canada;), which recognizes conserved epitopes within the gp63-family metalloproteases in their native state, followed by appropriate fluorescent secondary antibodies.

Techniques: Immunofluorescence, Microscopy, Staining, Labeling, Membrane, Confocal Microscopy, Imaging

Journal: Pathogens

Article Title: Acanthamoeba castellanii : Non-Steroidal Anti-Inflammatory Drugs Affect Adhesion, Motility, and Encystment, Suggesting a Link with a gp63-like Protein Candidate

doi: 10.3390/pathogens15030263

Figure Lengend Snippet: Comparative domain architecture and structural analysis of gp63 metalloproteases from Acanthamoeba castellanii and Leishmania major . ( A , C ) gp63 from A. castellanii ; ( B , D ) gp63 from L. major . ( A , B ) Schematic representation of gp63 domain organization showing predicted signal peptide (orange), activating proregion (pink), and catalytic peptidase M8 domain (aquamarine); sites of glycosylation (GlcNAc), metal-binding (Zn 2+ ), and other post-translational modifications are also pointed out. The theoretical molecular weight (kDa) and isoelectric point (pI) are indicated for each protein. ( C , D ) Structural validation of the predicted 3D folding models. ( Upper left ) Ramachandran plots with most residues in favored regions and few outliers (pink circles). ( Upper right ) 3D ribbon models of gp63 colored by confidence score (pLDDT). ( Bottom ) B-factor plots illustrating residue flexibility and secondary structure assignment.

Article Snippet: Samples were incubated with a commercial monoclonal anti- Leishmania major gp63 antibody (anti- Leishmania major monoclonal antibody (Cat. No. CLP005A, (CEDARLANE Laboratories Limited, Burlington, ON, Canada;), which recognizes conserved epitopes within the gp63-family metalloproteases in their native state, followed by appropriate fluorescent secondary antibodies.

Techniques: Glycoproteomics, Binding Assay, Molecular Weight, Biomarker Discovery, Residue

Journal: Pathogens

Article Title: Acanthamoeba castellanii : Non-Steroidal Anti-Inflammatory Drugs Affect Adhesion, Motility, and Encystment, Suggesting a Link with a gp63-like Protein Candidate

doi: 10.3390/pathogens15030263

Figure Lengend Snippet: Predicted binding interactions of nonsteroidal anti-inflammatory drugs (NSAIDs) with gp63 metalloproteases from A. castellanii and L. major . ( A , C , E ) gp63 of A. castellanii ; ( B , D , F ) gp63 of L. major . Docking interactions of diclofenac ( A , B ); ibuprofen ( C , D ); aspirin ( E , F ). Protein residues are shown in blue and ligands in orange. Yellow dashed lines highlight metal-complex interactions involving Zn 2+ ; and gray dashed lines denote hydrophobic contacts. Residues involved in ligand stabilization are detailed in 2D maps (black squares). Two-dimensional interaction diagrams for diclofenac, ibuprofen, and aspirin, showing the specific amino acid residues involved in ligand binding. The interactions include van der Waals forces, conventional hydrogen bonds in green dashed lines, and coordination with the catalytic metal ion in red lines.

Article Snippet: Samples were incubated with a commercial monoclonal anti- Leishmania major gp63 antibody (anti- Leishmania major monoclonal antibody (Cat. No. CLP005A, (CEDARLANE Laboratories Limited, Burlington, ON, Canada;), which recognizes conserved epitopes within the gp63-family metalloproteases in their native state, followed by appropriate fluorescent secondary antibodies.

Techniques: Binding Assay, Ligand Binding Assay

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) UMAP visualization of mouse hippocampal cell clusters classified by cell type based on DEG identified by Seurat v4. Violin plots represent the log-normalized expression of Itm2b and Trem2 across cell populations in mouse hippocampal cell clusters. ( B ) Human DFC cell clusters, classified by cell type as in ( A ). Violin plots represent the log-normalized expression of ITM2B and TREM2 across cell populations in human DFC cell clusters. ( C ) List of cell-type- specific marker genes used to annotate major brain populations. ( D ) Itm2b and Trem2 mRNA expression in mouse microglia and non-microglia cells analyzed by quantitative RT-PCR. Data information: The data sets analyzed are publicly available and are described in the two following papers: mouse scRNAseq data set (Zeisel et al, ), hippocampus n = 5 females, n = 5 males; human snRNAseq data set (Li et al, ), n = 10 (sex not specified). More information about these datasets can be found in the cited paper. Statistical comparisons between the groups shown in ( D ) was conducted using two-tailed unpaired t test **** P < 0.0001. The data are derived from are from 15-month-old w/w control animal, male n = 3, females n = 3; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques: Expressing, Marker, Quantitative RT-PCR, Two Tailed Test, Derivative Assay, Control

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) UMAPs of objects Data 1 and Data 2 before filtering. ( B ) UMAPs of objects Data 1 and Data 2 after filtering. ( C ) UMAP of Object 1 after integration, filtering, and re-clustering, shows 16 microglia clusters. Data information: The data presented in this analysis are the result of two experiments, namely Data 1 and Data 2. To combine specific sample datasets from both Data 1 and Data 2, we employed the integration feature within the Seurat package. By utilizing the first 20 principal components, we integrated these datasets into a single entity referred to as “Object1,” which encapsulates information from a total of 297,215 cells. These cells derive from: Trem2-KO , 1 male and 1 female; Itm2b-KO , 2 males and 2 females; WT controls, 1 male and 2 females; Itm2b/Trem2-dKO , 1 male and 1 female. The scRNAseq data are deposited at https://www.ncbi.nlm.nih.gov/geo/info/seq.html , GSE233601 to allow public access once the data are published.

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques:

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) UMAPs of microglia grouped by genotype. ( B ) Average scaled expression levels of selected signature genes per cluster and cluster’s annotation based on expression of signature genes. ( C ) Volcano plots showing differentially expressed genes in clusters I/T-D1, 2, 3 and 4. ( D ) Proportional contribution of each genotype and proportional contribution of individual samples of each genotype to cluster 3. ( E ) KEGG pathway enrichment analysis of pathways upregulated in cluster 3. Data information: The data presented in this analysis are the result of two experiments, namely Data 1 and Data 2. To combine specific sample datasets from both Data 1 and Data 2, we employed the integration feature within the Seurat package. By utilizing the first 20 principal components, we integrated these datasets into a single entity referred to as “Object1,” which encapsulates information from a total of 297,215 cells. Volcano plots in ( C ) were obtained using Fast Wilcoxon rank sum test and auROC. These cells derive from: Trem2-KO , n = 1 male and n = 1 female; Itm2b-KO , n = 2 males and n = 2 females; WT controls, n = 1 male and n = 2 females; Itm2b/Trem2-dKO , n = 1 male and n = 1 female. The scRNAseq data are deposited at https://www.ncbi.nlm.nih.gov/geo/info/seq.html , GSE233601 to allow public access once the data are published. All data are expressed as means +/− SEM.

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques: Expressing

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) UMAPs of re-clustered microglia in Object 1. ( B ) UMAPs split by individual samples. ( C ) Average scaled expression levels of selected signature genes per cluster and cluster’s annotation based on expression of signature genes. ( D ) Proportional contribution of each genotype to each cluster. Cluster 3 was highly represented in Itm2b-KO mice, with 89% of microglia in this cluster originating from these mice. Conversely, Cluster 7 was preponderant in WT controls. However, ~93% of the cells assigned to cluster 7 derived from one WT control animal (the male WT control, as depicted in UMAP plot b). Therefore, the observed expansion of cluster 7 is attributed to animal-specific factors rather than genotype-specific factors. ( E ) Gene expression heatmap showing the top 5 enriched genes for each microglia cluster. The number of cells per cluster is denoted above the cluster. Itm2b is one of the top genes downregulated in cluster 3 because 89% of the cells in this cluster are from Itm2b-KO mice. Enlarged heatmap of Clusters 8 to 15 is also show (right) for better visibility. Data information: The data presented in this analysis are deposited at https://www.ncbi.nlm.nih.gov/geo/info/seq.html , GSE233601 to allow public access once the data are published.

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques: Expressing, Derivative Assay, Control, Gene Expression

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Schematic representation of ELISA 1 and ELISA 2. Both ELISAs use the same Biotinylated-αTrem2 capture antibody (in black). ELISA 1 uses αTrem2-CT (red) + Sulfo-αRabbit (blue) detection antibodies. ELISA 2 uses αTrem2-NT (orange) + Sulfo-αRat (green) detection antibodies. Trem2 can be detected by both ELISAs, sTrem2 can be detected only by ELISA 2: neither ELISA can detect Trem2-CTF. ( B ) Quantification of Trem2 and sTrem2 in the P100 and S100 brain fractions of ~245 days old w/w control, Itm2b-KO and Trem2-KO mice. ( C ) Western blot analysis of P100 fractions from a representative w/w, Trem2-KO and Itm2b-KO P100 sample with αTrem2-CT and an αBri2 antibody. *Indicates a non-specific band. ( D ) Detection and quantification of Trem2-CTF in the P100 fraction by Western blot analysis and with Image Lab software; GAPDH was used as a loading control. ( E ) ELISA measurements of endogenous Aβ40 and Aβ42 in brain homogenates of w/w, Trem2-KO and Itm2b-KO animals. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. The membrane in ( C ) was cut at the 20 and 15 kDa molecular weight marker (MWM). The upper section was probed with the anti-Bri2 antibody, while the lower section was probed with the Trem2-CT antibody. Similarly, in ( D ), the two membranes were divided at the 20 and 15 kDa MWM. The upper portion was probed with the anti-Gapdh antibody, while the lower portion was probed with the Trem2-CT antibody. Statistical comparisons among the groups were conducted using one-way ANOVA followed by post-hoc Tukey’s multiple comparisons test when ANOVA showed significant differences ( B , E ); two–way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA showed significant differences ( D ). ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data presented are derived from are from w/w control, females n = 7, males n = 12; Itm2b-KO females n = 6, males n = 7; Trem2-KO , females n = 6, males n = 7; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot, Software, Membrane, Molecular Weight, Marker, Derivative Assay

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) CD11b and CD45 staining, and FACS analysis of brain cells isolated from Cx3cr1 CreER/wt and Cx3cr1 wt/wt animals. ( B ) FACS analysis of sorted EYFP + (microglia) and EYFP - (non-microglia) brain cell populations from Itm2b f/f :Cx3cr1 CreER/wt animals. ( C ) Schematic representation of the PCR test used to identify the Itm2b f and Itm2b KO alleles. ( D ) PCR analysis of genomic DNA isolated from EYFP + and EYFP - cells sorted from Itm2b f/f :Cx3cr1 CreER/wt brains. ( E ) Analysis of Itm2b and Trem2 mRNA expression in sorted EYFP + (microglia) and EYFP - (non-microglia) brain cell populations from ~14 months-old Itm2b f/f :Cx3cr1 CreER/wt and Itm2b w/w :Cx3cr1 CreER/wt animals. ( F ) ELISA 2 was used to measure sTrem2 levels in Itm2b f/f :Cx3cr1 CreER/wt and Itm2b f/f :Cx3cr1 wt/wt littermates. Data information: Statistical comparisons among the groups were conducted two-way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA showed significant differences ( E ); two-tailed unpaired t test ( F ). ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data presented are derived from: (E) Itm2b f/f :Cx3cr1 CreER/wt , females n = 5, males n = 7; Itm2b w/w :Cx3cr1 CreER/wt , females n = 3, males n = 4; ( F ) Itm2b f/f :Cx3cr1 CreER/wt , females n = 15, males n = 12; Itm2b f/f :Cx3cr1 wt/wt , females n = 10, males n = 11; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques: Staining, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Derivative Assay

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Quantification of sTrem2 in the conditioned media of WT and Itm2b-KO primary microglia using ELISA (left panel) and Western blot of deglycosylated conditioned media with Trem2 NT antibody (quantification of Western blot is shown in the second panel). Trem2 CT antibody does not show any signal. ( B ) Quantification of sTrem2 in the conditioned media of WT and Itm2b-KO primary microglia after 5 h of serum starvation by ELISA. ( C ) Quantification of sTrem2 in the conditioned media of WT and Itm2b-KO primary microglia after 1 and 2 h with either vehicle (Veh) or E. coli by ELISA. ( D ) Western blot of deglycosylated cell lysates from WT and Itm2b-KO primary microglia, 2 h after E. coli stimulation, with Trem2 CT antibody to visualize Trem2 f.l. and Trem2-CTF, along with quantification of the Trem2-CTF/Trem2 f.l. ratio. ( E ) Quantification of the Trem2-CTF/Trem2 f.l. ratio for only the two untreated (Veh) groups. ( F ) Analysis of Itm2b and Trem2 mRNA expression in WT and Itm2b-KO primary microglia using quantitative RT-PCR. ( G ) A second set of biological replicates was analyzed following the same procedure as in panel ( C ). ( H ) A second set of biological replicates was analyzed following the same procedure as in panel ( D ). ( I ) A second set of biological replicates was analyzed following the same procedure as in panel ( F ). Data information: Statistical comparisons among the groups were conducted using either a two-tailed unpaired t -test ( A , B , E , F , I ) or a two-way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA indicated significant differences ( C , D , G , H ). * P < 0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001. The data presented are derived from WT primary microglia cultures ( n = 3 for ( A , C , D , E – I ), n = 6 for ( B )) and Itm2b-KO primary microglia ( n = 3 for Exp. and n = 3 for ( A , C , D , E – I ), n = 6 for ( B )); the letter “n” indicates biological replicates, except for ( B ), which includes 3 biological replicates with 2 technical replicates each. Each biological replicate was composed of primary microglia generated from 2 P2 pups. All data are expressed as means +/− SEM. .

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Quantitative RT-PCR, Two Tailed Test, Derivative Assay, Generated

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Western blot analysis with Trem2 CT antibody of deglycosylated cell lysates from Itm2b-KO microglia treated with either vehicle (PBS) or a 2 μM concentration of BRI2-ECD. Quantification of the Trem2-CTF/Trem2 f.l. ratios from the Western blot shown in the right panel. ( B ) sTrem2 ELISA on conditioned media from these cell cultures (left panel). The right panel shows an ELISA performed using media from cells treated with vehicle and incubated before and during the ELISA with either vehicle (PBS) or 2 μM of BRI2-ECD. The evidence that incubation with BRI2-ECD does not change the ELISA quantification indicates that BRI2-ECD does not interfere with the quantification of sTrem2 by ELISA. ( C ) Western blot analysis with anti-Syk and anti-pSyk antibodies of cell lysates from Itm2b-KO microglia treated with either vehicle (PBS) or a 2 μM concentration of BRI2-ECD. Quantification of the pSyk/Syk ratios from the Western blot is shown in the right panel. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals. Statistical comparisons among the groups were conducted using a two-tailed unpaired t -test. * P < 0.05, ** P < 0.01, **** P < 0.0001. The data presented are derived from Itm2b-KO primary microglia ( n = 3 for each condition); the letter “n” indicates biological replicates. Each biological replicate was composed of primary microglia generated from 2 P2 pups. All data are expressed as means +/− SEM. .

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques: Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Two Tailed Test, Derivative Assay, Generated

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Quantification of Adam10 and Adam17 mRNA expression in WT and Itm2b-KO primary microglia using quantitative RT-PCR. ( B ) The experiment is a replicate of the one described in ( A ), using separate biological replicates. ( C ) Quantification of Adam17 in cell lysates from WT and Itm2b-KO primary microglia taken 2 h post E. coli stimulation (including controls treated solely with vehicle, Veh). The Western blot corresponding to the quantification is shown to the right and was performed using the membrane probed with α-pSyk, α-pPlcγ1, and α-p-p38 antibodies in Fig. . ( D ) The experiment is a replicate of the one described in ( C ), using separate biological replicates. The Western blot corresponding to the quantification is shown to the right and was performed using the membrane probed with α-pSyk, α-pPlcγ1, and α-p-p38 antibodies in Fig. . ( E ) ELISA-based quantification of TNFα secretion in the culture supernatants of WT and Itm2b-KO primary microglia treated with E. coli for 1 h, including controls treated solely with vehicle (Veh). ( F ) The experiment is a replicate of the one described in ( E ), using separate biological replicates. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals. Statistical comparisons among the groups were conducted using either a two-tailed unpaired t -test ( A , B ) or a two-way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA indicated significant differences ( C – F ). * P < 0.05, *** P < 0.001, **** P < 0.0001. The data presented are derived from WT primary microglia cultures ( n = 3) and Itm2b-KO primary microglia ( n = 3); the letter “n” indicates biological replicates. Each biological replicate was composed of primary microglia generated from 2 P2 pups. All data are expressed as means +/− SEM. .

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Membrane, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Derivative Assay, Generated

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Western blot of cell lysates from both WT and Itm2b-KO primary microglia, taken 2 h post E. coli stimulation (along with controls treated solely with vehicle, Veh), for the detection of Syk, pSyk, Plcγ1, pPlcγ1, p38, and p-p38. Two membranes were employed for analysis. The first membrane was initially probed with an α-Syk antibody, followed by an α-Plcγ1 antibody, and subsequently with an α-p38 antibody. On the other hand, the second membrane was initially probed with an α-pSyk antibody, followed by an α-pPlcγ1 antibody, and concluded with an α-p-p38 antibody. *Indicates a non-specific band. ( B ) Quantification of Western blots from ( A ), depicting the ratios of pSyk/Syk, pPlcγ1/Plcγ1, and p-p38/p38, which reflect the activation levels of Syk, Plcγ1, and p38, respectively. ( C ) The experiment is a replicate of the one described in ( A ), using separate biological replicates. ( D ) Quantification of Western blots shown in ( C ). Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals. Statistical comparisons among the groups were conducted using a two-way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA indicated significant differences. * P < 0.05, *** P < 0.001, **** P < 0.0001. The data presented are derived from WT primary microglia cultures ( n = 3) and Itm2b-KO primary microglia ( n = 3); the letter “n” indicates biological replicates. Each biological replicate was composed of primary microglia generated from 2 P2 pups. All data are expressed as means +/− SEM. .

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques: Western Blot, Membrane, Activation Assay, Derivative Assay, Generated

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A – D ) WT and Itm2b-KO primary microglia were incubated with pHrodo Red E . coli BioParticles and the kinetics of phagocytosis was measured in the IncuCyte imaging platform at 20 min intervals. Four independent experiments are shown. Data information: Statistical comparisons between genotypes were conducted using two-tailed unpaired t -test. **** P < 0.0001. The data presented are derived from WT primary microglia cultures ( n = 3 for ( A , B ), n = 4 for ( C , D )) and Itm2b-KO primary microglia ( n = 3 for ( A , B ), n = 4 for ( C , D )); the letter “n” indicates biological replicates. Four images (equivalent to 4 technical replicates) were captured for each sample at every time point. Each biological replicate was composed of primary microglia generated from 2 P2 pups. All data are expressed as means +/− SEM. .

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques: Incubation, Imaging, Two Tailed Test, Derivative Assay, Generated

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Quantification of IL-10, IL-16, IL-1β, IL-6, Cxcl-1, MCP-1, MIP-1α, MIP-2, MIP-3, and TNFα, in the conditioned media of WT and Itm2b-KO primary microglia treated with E. coli for 2 h, along with controls treated with vehicle, Veh. ( B ) Quantification of IL-6, Cxcl-1, MIP-1α, and TNFα secreted by WT and Itm2b-KO primary microglia grown for 24 h in complete media and after 5 h of serum starvation. Data information: Statistical comparisons among the groups were conducted using either two-way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA indicated significant differences ( A ), or two-tailed unpaired t -test ( B ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data presented are derived from: ( A ) WT primary microglia cultures ( n = 3 for Exp. 1 and n = 3 for Exp. 2) and Itm2b-KO primary microglia ( n = 3 for Exp. 1 and n = 3 for Exp. 2); ( B ) WT primary microglia cultures ( n = 6) and Itm2b-KO primary microglia ( n = 6); the letter “n” indicates biological replicates, except for ( B ), which includes 3 biological replicates with 2 technical replicates each. Each biological replicate was composed of primary microglia generated from 2 P2 pups. All data are expressed as means +/− SEM. .

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques: Two Tailed Test, Derivative Assay, Generated

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: Reagents and tools.

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Sequencing, Gene Expression, Blocking Assay, Western Blot, Isolation, Biomarker Discovery, Software, Imaging, Real-time Polymerase Chain Reaction

Journal: Blood

Article Title: Differential regulation of CXCR2 trafficking by Rab GTPases

doi: 10.1182/blood-2002-07-1965

Figure Lengend Snippet: For the specific staining of CXCR2, cells stably expressing (A) or not expressing (B) CXCR2 were fixed in methanol. Cells were incubated with a monoclonal CXCR2 antibody for 30 minutes, followed by incubation with an FITC-conjugated antimouse antibody for 30 minutes. Specific cell surface expression of CXCR2 was determined by confocal microscopy. For the colocalization of CXCR2 with the endogenous Rab proteins, cells stably expressing CXCR2 were treated with CXCL8 (200 nM) for 1 hour (C–F) or 4 hours (G–H), then fixed in methanol. Cells were incubated with a mixture of a monoclonal CXCR2 antibody and a rabbit antibody for Rab5 (C–D), Rab11a (E–F), or Rab7 (G–H), for 30 minutes. After washing with PBS, cells were incubated with a mixture of an FITC-conjugated antimouse antibody and a rhodamine-conjugated antirabbit anti-body for 30 minutes. Representative confocal micrographs from 3 independent experiments demonstrating the intracellular localization of CXCR2 (C,E,G), Rab5 (D), Rab11a (F), and Rab7 (H) are shown. Arrows indicate the colocalization of CXCR2 with the individual Rab proteins. Bars, 10 μm.

Article Snippet: Cells were washed and incubated with an antibody mixture containing a rhodamine-conjugated antirabbit antibody (Molecular Probes, Eugene, OR) and a fluorescein isothiocyanate (FITC)-conjugated antimouse antibody (Molecular Probes) for 30 minutes.

Techniques: Staining, Stable Transfection, Expressing, Incubation, Confocal Microscopy

Journal: Blood

Article Title: Differential regulation of CXCR2 trafficking by Rab GTPases

doi: 10.1182/blood-2002-07-1965

Figure Lengend Snippet: Cells stably expressing CXCR2 were incubated with CXCL8 (200 nM) and transferrin–Texas Red (25 μg/mL) (A–B) or Dil-LDL (40 μg/mL) (C, D) at 37°C for 1 hour (A, C) or 4 hours (B, D). Cells were washed in cold PBS and fixed in methanol. Cells were incubated with a monoclonal CXCR2 antibody for 30 minutes at room temperature, followed by incubation with an FITC-conjugated antimouse antibody for 30 minutes at room temperature. Representative laser-scanning confocal micrographs from 3 independent experiments demonstrating the distribution of CXCR2 (green), transferrin–Texas Red (red), Dil-LDL (red), and colocalization of CXCR2 with transferrin–Texas Red (yellow; A–B) or CXCR2 with Dil-LDL (yellow; C–D) are shown. Bars, 10 μm. (E) Quantification of the percentage of CXCR2 colocalized with transferrin (0) or LDL (f) was determined by counting the colocalized receptor fluorescence (yellow) from the total internalized receptor fluorescence (green and yellow). Data are means ± SEMs of 3 independent experiments (**P < .01). Tf indicates transferrin.

Article Snippet: Cells were washed and incubated with an antibody mixture containing a rhodamine-conjugated antirabbit antibody (Molecular Probes, Eugene, OR) and a fluorescein isothiocyanate (FITC)-conjugated antimouse antibody (Molecular Probes) for 30 minutes.

Techniques: Stable Transfection, Expressing, Incubation, Fluorescence

Journal: Blood

Article Title: Differential regulation of CXCR2 trafficking by Rab GTPases

doi: 10.1182/blood-2002-07-1965

Figure Lengend Snippet: HEK293 cells stably expressing CXCR2 were transiently transfected with plasmids for vector (A–C) Rab11a (D–F) or Rab11a-S25N (G–I). Cells were treated with CXCL8 at 37°C for 30 minutes, then the agonist was removed and the cells were recovered by incubation with agonist-free medium for 0 minutes (A,D,G), 30 minutes (B,E,H), or 1 hour (C,F,I) at 37°C. For the staining of the cell surface receptor, cells were incubated with a monoclonal CXCR2 antibody at 4°C for 1 hour, followed by incubation with FITC-conjugated antimouse IgG at 4°C for 30 minutes. Cells were washed and fixed in 2% formaldehyde in PBS and analyzed in FACScan. Thin solid line represents the staining of cells in the absence of primary CXCR2 antibody (background). Thick solid line represents the immunostaining of cell surface CXCR2 in the cells without CXCL8 treatment (total). Broken line indicates the immunostaining cell surface CXCR2 in the cells treated with CXCL8 and recovered for different intervals. (J) Relative cell surface fluorescence (percentage of total) of the cells recovered for different intervals was quantified. Data are means ± SEMs of 4 independent experiments (*P < .05).

Article Snippet: Cells were washed and incubated with an antibody mixture containing a rhodamine-conjugated antirabbit antibody (Molecular Probes, Eugene, OR) and a fluorescein isothiocyanate (FITC)-conjugated antimouse antibody (Molecular Probes) for 30 minutes.

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Incubation, Staining, Cell Surface Receptor Assay, Immunostaining, Fluorescence

Journal: Blood

Article Title: Differential regulation of CXCR2 trafficking by Rab GTPases

doi: 10.1182/blood-2002-07-1965

Figure Lengend Snippet: (A–C) Colocalization of CXCR2 with LAMP-1. HEK293 cells stably cotransfected with plasmids encoding CXCR2 and vector (A), CXCR2 and Rab7 (B), or CXCR2 and Rab7-T22N (C) were treated with CXCL8 at 37°C for 4 hours. Cells were fixed with methanol and incubated with a mixture of a rabbit CXCR2 antibody and a mouse monoclonal Lamp-1 antibody for 30 minutes, followed by a mixture of an FITC-conjugated antirabbit antibody and a rhodamine-conjugated antimouse antibody for 30 minutes. Representative laser-scanning confocal micrographs from independent experiments demonstrating the distribution of CXCR2 (green) and Lamp-1 (red) and the colocalization of CXCR2 with Lamp-1 (yellow) are shown. (D–E) Colocalization of LDL with LAMP-1. HEK293 cells stably expressing Rab7 (D) or Rab7-T22N (E) were incubated with Dil-LDL (40 μg/mL) at 37°C for 4 hours, then fixed in methanol. Cells were incubated with a monoclonal LAMP-1 antibody followed by incubation with an FITC-conjugated antimouse anti-body. Representative confocal micrographs from 3 independent experiments demonstrating the distribution of LDL (red), Lamp-1 (green), and the colocalization of LDL with Lamp-1 (yellow) are shown. Images were processed using Photoshop software. Bars, 10 μm.

Article Snippet: Cells were washed and incubated with an antibody mixture containing a rhodamine-conjugated antirabbit antibody (Molecular Probes, Eugene, OR) and a fluorescein isothiocyanate (FITC)-conjugated antimouse antibody (Molecular Probes) for 30 minutes.

Techniques: Stable Transfection, Plasmid Preparation, Incubation, Expressing, Software