mouse anti-timp3  (Absolute Biotech Inc)

Journal: The Journal of Immunology Author Choice

Article Title: The Metalloproteinase ADAMTS5 Is Expressed by Interstitial Inflammatory Cells in IgA Nephropathy and Is Proteolytically Active on the Kidney Matrix

doi: 10.4049/jimmunol.2000448

Figure Lengend Snippet: Identification of matrix proteins by urine proteomics leads to the discovery of ADAMTS5 in IgAN kidneys. ( A ) LC-MS/MS proteomics of healthy donor and IgAN patient urine identified multiple ECM proteins present in IgAN urine (see Ref. ). Matrix proteins were selected using gene ontology (BinGO) and are presented as a protein-protein interaction network. Yellow and red proteins are increased in IgAN urine, gray are unchanged, and blue are decreased in comparison with healthy samples. Proteomics analysis including all identified proteins is described in detail in . ( B ) The mRNA expression of ADAMTS5 and TIMP3 was examined in a published microarray dataset comparing 25 IgAN patients with six healthy donor kidney biopsies. Microarray gene expression was retrieved directly from National Center for Biotechnology Information–Gene Expression Omnibus (GSE35488; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35488 ). Microarray data are available in . ADAMTS5 and TIMP3 were significantly regulated in IgAN . Differences were examined using standard t test using data curated and processed by NCBI-GEO as described in GSE35488; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35488 . The p values are shown. ( C ) ADAMTS5 protein immunofluorescence was measured in 13 healthy (kidney transplant donors) versus 20 IgAN patients. IgAN donors were biopsy diagnosed. ADAMTS5 staining was quantified using ADAMTS5-stained particle counting corrected to biopsy tissue area on ImageJ and values were compared using two-tailed t test. ( D ) For the 20 IgAN patients, ADAMTS5 immunofluorescence was correlated to their matching urine protein concentration (proteinuria; mg/dl) and eGFR (estimated glomerular filtration rate; ml/min/1.73 m 2 ) values. The r and p values were computed using nonparametric Spearman test given the dissimilar nature of the correlated values. ( E ) ADAMTS5 staining was also compared in the same IgAN biopsies but biopsies were grouped according to their histological MEST-C score. We focused on the (T) score (percentage of biopsy area affected by tubular atrophy or interstitial fibrosis, whichever is greater) and compared less affected T0 biopsies ( n = 7) with more affected T1/T2 biopsies ( n = 13). There was a significant increase in ADAMTS5 staining in T1/T2 specimens ( t test). ( F ) ADAMTS5 biopsy staining was also measured separately in the tubulointerstitium and glomeruli and compared in healthy versus IgAN biopsies. ADAMTS5 staining in the IgAN interstitium ( IgAN Interst ) is significantly increased in comparison with staining in IgAN glomeruli ( IgAN Glom ), as well as in comparison with healthy interstitium and glomeruli, which are NS different to each other (ANOVA with Fisher least significant difference, multiple comparison test). ( G and H ) Examples of ADAMTS5 immunofluorescence in IgAN and healthy kidney biopsies costained with relevant proteins. In (G) ADAMTS5 (red) was costained with ADAMTS1 (green) and TIMP3 (blue). TIMP3 staining was below the threshold of confident detection in either IgAN or healthy biopsies. In (H) ADAMTS5 (red) was costained with TIMP1 (green). ADAMTS5 is close but does not appear to colocalise with TIMP1 in affected tubulointerstitial areas. tub denotes renal tubules and glom denotes glomeruli. Note that ADAMTS5 is increased in areas of tubulointerstitial inflammatory infiltration and remodelling. IgAN glomeruli also contain ADAMTS5 + cells. In healthy biopsies, there were sporadic ADAMTS5-stained (red) cells in the interstitium [(G) healthy] and glomeruli [(H) healthy]. White-dashed boxes denote areas shown in higher magnification in lower panels.

Article Snippet: Sections were treated using acid-based Ag retrieval and dewaxing solution (Abcam) at 98°C for 1 h. Sections were thoroughly washed, blocked in 10% normal donkey serum (Dako) for 1 h, and subsequently incubated with anti-human primary Abs against the following proteins: ADAMTS5 (raised in rabbit), CD64, and vimentin (mouse) from Abcam and TIMP3 (mouse), myeloperoxidase, TIMP1, lumican (LUM), ADAMTS1, versican (VCAN), collagen-4, and CD206 (all goat) from Bio-Techne.

Techniques: Liquid Chromatography with Mass Spectroscopy, Comparison, Expressing, Microarray, Immunofluorescence, Staining, Two Tailed Test, Protein Concentration, Filtration

Journal: bioRxiv

Article Title: Regulatory mechanisms in multiple vascular diseases locus LRP1 involve repression by SNAIL and extracellular matrix remodeling

doi: 10.1101/2023.05.09.539992

Figure Lengend Snippet: A : Illustration of indel frameshift in exons of LRP1 to generate knockout iPSCs using CRISPR/Cas9 prior to differentiation into SMCs B: Representative Western blot images showing LRP1 protein expression in WT and LRP1 KO iPSC-SMCs. C : Heatmap representation of relative RNA expression for differentially expressed genes between LRP1 KO and WT iPSC-SMCs. Genes involved in “collagen containing extracellular matrix” (gene ontology 0062023) are indicated D : Representative Western blot images showing the expression of phospho-SMAD2/3 (p-SMAD2/3) and total SMAD2/3 (SMAD2/3) in WT and LRP1 KO iPSC-SMCs in the presence of TGFβ1 protein for 1 hour. E : Heatmap representation of label free quantification (LFQ) for proteins over or underrepresented in extracellular extracts of LRP1 KO and WT iPSC-SMCs. F : Representative Western blot images showing the expression of LRP1, CYR61 and TIMP3 in whole cell extracts or decellularized extracts (ECM).

Article Snippet: Following antibodies were used in Western blot experiments: rabbit anti-LRP1 (ab92544, Abcam, Cambridge, UK), mouse anti-β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, Texas, USA), rabbit anti-phospho-SMAD2/3 (#8828) and anti-SMAD2/3 (#3102, Cell Signaling Technology, Danvers, Massachusetts, USA), mouse anti-TIMP3 (LS-B2576, Lifespan Biosciences, Seattle, Washington, USA), mouse anti-CYR61 (sc-374129, Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Knock-Out, CRISPR, Western Blot, Expressing, RNA Expression

Journal: Developmental cell

Article Title: Pericyte ALK5/TIMP3 axis contributes to endothelial morphogenesis in the developing brain

doi: 10.1016/j.devcel.2018.01.018

Figure Lengend Snippet: Key resources table

Article Snippet: Primary antibodies used for Western blot analysis were rabbit anti-ALK5 (Abcam, ab31013), rabbit anti-phospho-SMAD2 (Cell Signaling, 3108), rabbit anti-phospho-SMAD3 (Abcam, ab52903), rabbit anti-SMAD2/3 (Cell Signaling, 3102), rabbit anti-GAPDH (Cell Signaling, 2118) and mouse anti-TIMP3 (Millipore, MAB3318).

Techniques: Plasmid Preparation, Blocking Assay, Virus, Recombinant, TA Cloning, Magnetic Beads, cDNA Synthesis, In Situ Hybridization

Journal: Developmental cell

Article Title: Pericyte ALK5/TIMP3 axis contributes to endothelial morphogenesis in the developing brain

doi: 10.1016/j.devcel.2018.01.018

Figure Lengend Snippet: (A) ALK5 and TIMP3 mRNA levels as assessed by qRT-PCR relative to GAPDH in Scr- and siALK5-treated human PCs and normalized to Scr levels (n=3). Student’s t test, ***p<0.001, ****p<0.0001, versus Scr. (B) PCs were treated with TGFβ1 for indicated times and RNA was isolated. Histogram shows TIMP3 mRNA levels relative to 18S rRNA as assessed by qRT-PCR and normalized to 0 hour (n=3). One-way ANOVA, *p<0.05 versus 0 hour. (C) Western blot of TIMP3 and GAPDH from co-cultures of PCs pretreated with Scr or siAlk5 and ECs (n=3). (D) Zs+ NG2+ CD31− cells (PCs) were isolated by FACS from the brains of E13.5 Pdgfrb-Cre, ROSA26R(Zs/+) embryos also carrying either Alk5(flox/+) (control) or Alk5(flox/flox) (mutant) as described in Figure S5. RNA levels of Alk5 and Timp3 relative to 18S rRNA were measured by qRT-PCR and normalized to control (n=3). Student’s t test, **p<0.01, ***p<0.001, versus control. (E) ECs co-cultured with Scr or siALK5 pretreated PCs on Matrigel in the presence of vehicle or rTIMP3. Representative Brightfield images are shown. Scale bar, 100 μm. (F to H) Histograms showing EC branches (F), branch points (G) and branch length (H) for rTIMP3- or vehicle-treated co-culture of ECs and Scr or siALK5-pretreated PCs normalized to vehicle-treated Scr PC + EC control (n=3 experiments, 6 fields per condition for each experiment). One-way ANOVA with Tukey’s post hoc test, **p<0.01, ***p<0.001. All data are averages ± SD. See also Figures S5–S7.

Article Snippet: mouse anti-TIMP3 , Millipore , Cat# MAB3318; RRID:AB_94813.

Techniques: Quantitative RT-PCR, Isolation, Western Blot, Mutagenesis, Cell Culture, Co-Culture Assay

Journal: Developmental cell

Article Title: Pericyte ALK5/TIMP3 axis contributes to endothelial morphogenesis in the developing brain

doi: 10.1016/j.devcel.2018.01.018

Figure Lengend Snippet: Key resources table

Article Snippet: mouse anti-TIMP3 , Millipore , Cat# MAB3318; RRID:AB_94813.

Techniques: Plasmid Preparation, Blocking Assay, Recombinant, TA Cloning, Magnetic Beads, In Situ Hybridization