lif neutralising antibody  (R&D Systems)

Journal: Nature Communications

Article Title: Macrophage-fibroblast JAK/STAT dependent crosstalk promotes liver metastatic outgrowth in pancreatic cancer

doi: 10.1038/s41467-024-47949-3

Figure Lengend Snippet: A UMAP plot depicting expression of Acta2 , Pdgfra , and Cd34 in vMAF, myMAF, iMAF, and cycMAF clusters. B Representative immunofluorescence image of annotated MAF subtypes in liver metastasis of KPC mice. Individual MAF subtypes are defined on the representative image in coloured regions: vMAF (αSMA Low , PDGFRα High , CD34 Low ) – red; myMAF (αSMA High , PDGFRα Low , CD34 Low ) – green; iMAF (αSMA Low , PDGFRα High , CD34 High ) – blue). Arrowheads indicate the defined MAF subtype. N = 3 mice. Scale bar, 50 µm. C Representative immunofluorescence image for the distribution of PDGFRα and and αSMA to discern vMAFs and myMAFs in metastatic tumours derived from Pdgfrb -GFP mice treated with IgG or αCSF1R neutralising antibody. To the right, separated channel images are shown. Scale bar, 50 µm. D Representative schematic depicting how the gradated expression of αSMA and PDGFRα separates across vMAF and myMAF subpopulations, as observed in A , is utilised as a strategy to analyse changes in abundance between both subpopulations. E Quantification of the distribution of αSMA low PDGFRα high and αSMA high PDGFRα low cells in C . Data is presented as mean percentage distribution averaged from n = 4 mice per group. Error bars, SD. P values, two-way ANOVA with Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

Article Snippet: LIF neutralising antibody (AF499, R&D Systems), or IgG control (AB-108-C, R&D Systems) was administered by intraperitoneal injection at 0.5 mg/Kg, diluted in PBS, starting from day 5, every 48 h, up until day 14.

Techniques: Expressing, Immunofluorescence, Derivative Assay

Journal: Acta Neuropathologica Communications

Article Title: Differential effects of mutations of POPDC proteins on heteromeric interaction and membrane trafficking

doi: 10.1186/s40478-022-01501-w

Figure Lengend Snippet: Two non-related patients carrying a BVES p.V183F variant and suffering from LGMDR25. a , b Axial muscle MRI images of a patient 1 (PT1) and b patient 2 (PT2). PT1 was scanned at age 17 displaying fatty replacement and hypotrophy of the gastrocnemius medialis (arrows), with hyperintensity on T2-STIR images (arrowheads). PT2 scanned at age 50 displaying, in addition to changes in the gastrocnemius medialis (arrows), also advanced fatty replacement of adductor longus greater than in adductor magnus (arrowheads) and diffuse T2-STIR hyperintense lesions in the thigh, more evident in the anterior compartment (asterisks). c – f HE stained transverse sections of muscle biopsies taken from c PT1, e PT2 and respective matched controls d CT1 and f CT2. Note the fiber size heterogeneity in both patients. Moderately hypertrophied (arrows) and hypo/atrophied muscle fibers (asterisks) are seen. In PT1, a prominent increase in connective tissue is present. g Sequence alignment of part of the Popeye domains of vertebrate and invertebrate POPDC proteins. Color code: V183 (yellow), conserved (turquoise) and similar (green) residues. h Model of the Popeye domain of POPDC1 with the position of V183 (cyan) and the mutant V183F (pink) residues highlighted. The position of the phenylalanine side chain was determined using the Dynameomics rotamer library . i The position of the V183F mutation relative to the predicted cAMP binding site as determined using the 3DLigandSite server . j A model showing the possible steric clashes between the side chain of V183F (pink) with other residues of the β-folds of the Popeye domain as predicted by the Dynameomics rotamer library

Article Snippet: POPDC1 was detected using a polyclonal goat anti-POPDC1 antibody (sc-49889, Santa Cruz Biotechnology).

Techniques: Variant Assay, Staining, Sequencing, Mutagenesis, Binding Assay

Journal: Acta Neuropathologica Communications

Article Title: Differential effects of mutations of POPDC proteins on heteromeric interaction and membrane trafficking

doi: 10.1186/s40478-022-01501-w

Figure Lengend Snippet: Membrane localization of POPDC isoforms in muscle fibers expressing the POPDC1 p.V183F variant. a and b Transverse sections of skeletal muscle biopsies from PT1 and PT2 harboring the POPDC1 p.V183F variant and respective matched controls (CT1 and CT2) were stained for a POPDC1 or b POPDC2 along with SGCA serving as a sarcolemma marker. Scale bar: 100 μm. c and d The expression levels of c POPDC1 and d POPDC2 at the sarcolemma were normalized to SGCA and quantified in individual fibers. The number of sections (sec), images (img) and fibers (fib) analyzed per group are as follows: CT1: POPDC1—2 sec, 5 img, 161 fib; POPDC2—2 sec, 5 img, 138 fib. PT1: POPDC1—1 sec, 4 img, 167 fib; POPDC2—1 sec, 4 img, 167 fibers. CT2: POPDC1—1 sec, 13 img, 681 fib; POPDC2—2 sec, 8 img, 339 fib. PT2: POPDC1—3 sec, 20 img, 835 fib; POPDC2—3 sec, 14 img, 453 fib. The median POPDC/SGCA-level in each control biopsy was set to 1. Dashed lines indicate the normalized median and interquartile range. Data were analyzed using Mann–Whitney test; **** p < 0.0001

Article Snippet: POPDC1 was detected using a polyclonal goat anti-POPDC1 antibody (sc-49889, Santa Cruz Biotechnology).

Techniques: Membrane, Expressing, Variant Assay, Staining, Marker, Control, MANN-WHITNEY

Journal: Acta Neuropathologica Communications

Article Title: Differential effects of mutations of POPDC proteins on heteromeric interaction and membrane trafficking

doi: 10.1186/s40478-022-01501-w

Figure Lengend Snippet: The expression of POPDC1 and POPDC2 is greatly reduced at the sarcolemma of skeletal muscle fibers expressing POPDC1 p.Q153X. a and b Transverse sections of skeletal muscle biopsies from a patient (PT) carrying the POPDC1 p.Q153X variant in homozygosity and a matched control (CT) were stained for a POPDC1 or b POPDC2, along with SGCA as a sarcolemma marker. Scale bar: 100 μm. c and d The expression levels of c POPDC1 and d POPDC2 in the sarcolemma normalized to SGCA, were quantified in individual fibers. The number of sections (sec), images (img) and fibers (fib) analyzed per group are as follows: CT: POPDC1—1 sec, 4 img, 238 fib; POPDC2—1 sec, 4 img, 163 fib. PT: POPDC1—1 sec, 3 img, 65 fib; POPDC2—1 sec, 3 img, 70 fib. The median POPDC/SGCA-level in each control biopsy was set to 1. Dashed lines indicate the normalized median and interquartile range. Data were analyzed using Mann–Whitney test; **** p < 0.0001

Article Snippet: POPDC1 was detected using a polyclonal goat anti-POPDC1 antibody (sc-49889, Santa Cruz Biotechnology).

Techniques: Expressing, Variant Assay, Control, Staining, Marker, MANN-WHITNEY

Journal: Acta Neuropathologica Communications

Article Title: Differential effects of mutations of POPDC proteins on heteromeric interaction and membrane trafficking

doi: 10.1186/s40478-022-01501-w

Figure Lengend Snippet: Intermediate reduction of POPDC1 and POPDC2 expression at the sarcolemma of skeletal muscle fibers of a homozygous Popdc2 W188X/W188X knockin mouse. a and b Transverse sections of the m. gastrocnemius of a 3-month-old homozygous Popdc2 W188X/W188X mouse mutant (W188X) and a wild-type control (WT) were stained for (A) POPDC1 or (B) POPDC2, along with SGCA as a sarcolemma marker. Scale bar: 100 μm. c and d The expression levels of c POPDC1 and d POPDC2 in the sarcolemma, normalized to SGCA, were quantified in individual fibers. The number of sections (sec), images (img) and fibers (fib) analyzed per group are as follows: WT: POPDC1—1 sec, 3 img, 164 fib; POPDC2—1 sec, 3 img, 143 fib. W188X: POPDC1—1 sec, 4 img, 95 fib; POPDC2—1 sec, 4 img, 93 fib. The median POPDC/SGCA-level in each control biopsy was set to 1. Dashed lines indicate the normalized median and interquartile range. The control and homozygous mutant pairs were compared using a Mann–Whitney test; **** p < 0.0001

Article Snippet: POPDC1 was detected using a polyclonal goat anti-POPDC1 antibody (sc-49889, Santa Cruz Biotechnology).

Techniques: Expressing, Knock-In, Mutagenesis, Control, Staining, Marker, MANN-WHITNEY

Journal: Acta Neuropathologica Communications

Article Title: Differential effects of mutations of POPDC proteins on heteromeric interaction and membrane trafficking

doi: 10.1186/s40478-022-01501-w

Figure Lengend Snippet: POPDC mutations have varying impacts on POPDC1 and POPDC2 sarcolemmal expression in skeletal muscle. Comparison of the fold change in POPDC1 and POPDC2 expression, normalized to SGCA, in the sarcolemma of skeletal muscle fibers from biopsy material of patients carrying the POPDC1 p.V183F and POPDC1 p.Q153X variants and a homozygous Popdc2 p.W188X knock-in mouse compared to matched controls or wild-type mouse. The total number of fibers analyzed are shown at the base of each bar. Data is displayed as median ± 95% CI. POPDC1 and POPDC2 values were compared using Kruskal–Wallis followed by Dunn’s multiple comparisons test; * p < 0.05; ** p < 0.01; **** p < 0.0001

Article Snippet: POPDC1 was detected using a polyclonal goat anti-POPDC1 antibody (sc-49889, Santa Cruz Biotechnology).

Techniques: Expressing, Comparison, Knock-In

Journal: Acta Neuropathologica Communications

Article Title: Differential effects of mutations of POPDC proteins on heteromeric interaction and membrane trafficking

doi: 10.1186/s40478-022-01501-w

Figure Lengend Snippet: Co-expression of POPDC1 and POPDC2 is required for plasma membrane localization in HEK293 cells. a POPDC1-ECFP, POPDC2-EYFP, or both were transiently expressed in HEK293 cells. The plasma membrane was marked using DiD. Scale bar: 10 μm. b Bar graph of the ratio of plasma membrane to cytoplasm expression of POPDC1 or POPDC2 (POPDC1-ECFP: n = 15, POPDC2-EYFP: n = 15, POPDC1-ECFP + POPDC2-EYP: n = 46; min., N ≥ 2). Bars show median ± 95% CI. The groups were compared using a Mann–Whitney test; **** p < 0.0001. c POPDC1 V183F-ECFP, Q153X-ECFP and S201F-ECFP and POPDC2 W188X-EYFP constructs were co-expressed with the appropriate wild-type POPDC partner in HEK293 cells. The plasma membrane was marked using DiD. Scale bar: 10 μm. d Bar graph of the ratio of plasma membrane to cytoplasm expression of POPDC1 or POPDC2 in the presence of the different POPDC1 and POPDC2 mutant proteins (WT n = 46, V183F n = 47, Q153X n = 17, S201F n = 22, W188X n = 24, min., N ≥ 2). Identical data are shown in b and d for the expression levels after co-transfection of both wild-type constructs. Bars show median ± 95% CI. Groups were compared using Kruskal–Wallis followed by Dunn’s test using the wild-type pair for comparison; **** p < 0.0001

Article Snippet: POPDC1 was detected using a polyclonal goat anti-POPDC1 antibody (sc-49889, Santa Cruz Biotechnology).

Techniques: Expressing, Clinical Proteomics, Membrane, MANN-WHITNEY, Construct, Mutagenesis, Cotransfection, Comparison

Journal: Acta Neuropathologica Communications

Article Title: Differential effects of mutations of POPDC proteins on heteromeric interaction and membrane trafficking

doi: 10.1186/s40478-022-01501-w

Figure Lengend Snippet: POPDC1 and POPDC2 undergo heteromeric complex formation. a Adult mouse ventricular cardiomyocytes immunostained for POPDC1 (red) and POPDC2 (green). b PLA of POPDC1 and POPDC2 in transverse sections of right atrium and left ventricle from wild-type and Popdc1 / Popdc2 knockout mice. Sections were counterstained with WGA (green) and DAPI. c and d Co-precipitation of c POPDC2-FLAG alone or together with POPDC1-MYC or POPDC3-MYC, respectively, or d POPDC1-HA alone or together with POPDC3-MYC. e Co-precipitation of ventricular tissue lysates of Popdc2 null mutant and wild-type mice. f Western blot of lysates from COS-7 cells expressing POPDC1-CFP and/or POPDC2-FLAG (left), or POPDC1-CFP and/or POPDC3-MYC (right). (x) monomer, (*) homodimer, (o) heterodimer (#) heterotetramer. g Quantitative Type-1 BRET saturation curves of POPDC1 and POPDC2 homo- and heteromeric complexes (N ≥ 2). h and i TREK-1 current in Xenopus oocytes expressing TREK-1 alone or together with POPDC1, POPDC2, or both. h Examples of TREK-1 current in response to a voltage jump from − 80 to 40 mV and i relative current amplitudes without or with theophylline (+ theo). Number of oocytes are given in each graph. Data are presented as mean ± SEM. * p < 0.05; *** p < 0.001. j and k Chimeric constructs containing j the N-terminal and transmembrane domains of POPDC1 and the cytoplasmic region of POPDC2 or k N-terminal and transmembrane domains of POPDC2 and the cytoplasmic region of POPDC1 were co-transfected with POPDC1 or POPDC3, respectively and subjected to co-precipitation analysis. l Truncations were introduced into POPDC1-MYC and subjected to co-precipitation analysis in COS-7 cells after co-transfection with POPDC2-FLAG. m A POPDC2-FLAG construct truncated to residue W188 was subjected to co-precipitation analysis after co-expression with POPDC1-MYC. n BiFC signal after co-expression of wild-type POPDC1 and POPDC2, or of POPDC1 p.V183F, p.Q153X, p.S201F and POPDC2 p.W188X mutants in HEK293 cells. POPDC1-VN155 + POPDC2-VN155 was used as a negative control. *** p < 0.001; **** p < 0.0001

Article Snippet: POPDC1 was detected using a polyclonal goat anti-POPDC1 antibody (sc-49889, Santa Cruz Biotechnology).

Techniques: Knock-Out, Mutagenesis, Western Blot, Expressing, Construct, Transfection, Cotransfection, Residue, Negative Control

Journal: Acta Neuropathologica Communications

Article Title: Differential effects of mutations of POPDC proteins on heteromeric interaction and membrane trafficking

doi: 10.1186/s40478-022-01501-w

Figure Lengend Snippet: The αC-helix of the Popeye domain mediates heteromeric complex formation between POPDC1 and POPDC2. a A model of heteromeric complex formation of the Popeye domains of POPDC1 (cyan) and POPDC2 (blue) using the structure of a CAP dimer (translucent; PDB: 1G6N) as a template. b Sequence alignment of the αC-helix of POPDC1, POPDC2 and POPDC3 from multiple vertebrate species and CAP protein from E. coli. A set of highly conserved hydrophobic amino acids are highlighted in red. c and d Overlay of the predicted αC-helical structures of POPDC1 (cyan), POPDC2 (blue) and POPDC3 (purple) with the side chains of the highly conserved hydrophobic residues. c Amino terminal and d side view. e and f The ratio of plasma membrane to cytoplasm expression levels of POPDC1-ECFP and POPDC2-EYFP in HEK293 cells, where either e POPDC1-ECFP or f POPDC2-EYFP was subjected to site-directed mutagenesis to introduce aspartic acid in place of a conserved hydrophobic residue within the αC-helix sequence. Total number of cells analyzed: POPDC1: L245D n = 71, F249D n = 56, I253D n = 27, I257D n = 40, L261D n = 47, L264D n = 42; POPDC2: I229D n = 48, F233D n = 20, L237D n = 45, I241D n = 42, L245D n = 63, L248D n = 56, N ≥ 2. Min. 2 transfections per group. Bars show median ± 95% CI. Groups were compared using Kruskal–Wallis followed by Dunn’s test using the wild-type pair as a comparison; ** p < 0.01, **** p < 0.0001. g and h Relationship between plasma membrane versus cytoplasm expression and each mutation in g POPDC1 and h POPDC2. i The predicted αC-helical Popeye domain interface between POPDC1 and POPDC2. Hydrophobic residues whose mutation to aspartic acid led to severely impaired plasma membrane localization of both POPDC proteins are labelled in red

Article Snippet: POPDC1 was detected using a polyclonal goat anti-POPDC1 antibody (sc-49889, Santa Cruz Biotechnology).

Techniques: Sequencing, Clinical Proteomics, Membrane, Expressing, Mutagenesis, Introduce, Residue, Transfection, Comparison