anti timp 1  (R&D Systems)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses.

doi: 10.1073/pnas.2306816121

Figure Lengend Snippet: Fig. 1. TIMP-1fl/fl knockout mice develop a severe EAE phenotype that is immunologically independent. (A) Schematic of design of development of the flox-targeted sites that enabled cell and tissue-specific deletion of TIMP-1 in GFAP-Cre-TIMP-1fl/fl mice. (B) Immunocytochemical validation of CRE expression in astrocytes from GFAP-Cre:TIMP-1fl/fl mice, and (C) physiological validation of TIMP-1 mRNA expression in primary astrocytes and absence of TIMP-1 mRNA expression in astrocyte cultures from GFAP-Cre:TIMP-1fl/fl mice (n = 8 per treatment; ANOVA; ****P < 0.0001; where *P < 0.01; ***P < 0.002). (D) Validation of absent TIMP-1 protein expression in primary astrocyte cultures from GFAP-Cre:Timp1fl/f mice (n = 3 biological replicates). (E) Clinical EAE in MOG35-55-treated C57Bl/6 WT and GFAP-Cre:TIMP-1fl/fl mice over 52 d time course (n = 13 to 17/treatment and genotype; 2-way ANOVA, P < 0.0001, where post-hoc multiple-comparisons identified significance at *P < 0.05 and **P < 0.01, as indicated). (F and G) Flow cytometry analysis of overall CD4+/IFNγ+ T cells from spinal cords of Wt and GFAP-Cre:TIMP- 1fl/fl mice at peak clinical EAE illness (day 18; n = 3 per genotype and treatment), including (H) analysis of CD4+/IFNγ+ T cells (t test, unpaired t test, P < 0.20).

Article Snippet: Anti- Fibronectin antibody (Biotin) (ab6584) (Abcam; rabbit, 1:5,000), Anti- Fibronectin antibody (ab23750) (Abcam; rabbit, 1:100- immunodepletion), Anti- Fibronectin antibody (ab2033) (Abcam; rabbit, 1:200), Anti- Timp1 antibody (AF980) (R&D Systems; goat, 1:125), Anti- MBP antibody (12) (Millipore; rat monoclonal, 1:500), Mouse anti- A2B5 (Invitrogen, Carlsbad, CA; 3 μg/mL), DAPI (Invitrogen, 1:1,000), Mouse anti- GFAP (Millipore, 1:400), Beta- actin (Sigma Aldrich, St. Louis, MO; mouse, 1:10,000).

Techniques: Knock-Out, Biomarker Discovery, Expressing, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses.

doi: 10.1073/pnas.2306816121

Figure Lengend Snippet: Fig. 2. Proteomic identification of elevated fibronectin (Fn) in secretome of TIMP-1KO astrocytes. (A and B) Topographic projections rendered using ProteomeLab™ of PF-2D analyses of extracellular proteins in secretome of primary astrocytes from WT (A and C) and TIMP-1KO (B and D) cultures. (C and D) Magnified view of differential peaks of interest identified by ProteomeLab™ which were interrogated by Liquid chromatography–mass spectrometry. (E) Western blot analysis of fibronectin (Fn) protein expression in ACM CM from WT and TIMP-1KO astrocytes. (F) Quantification of Fn from ACM from primary WT and TIMP-1KO astrocyte cultures showed TIMP-1KO astrocyte cultures had elevated Fn protein levels that were reduced with the addition of rmTIMP-1. (G) Analysis of Fn mRNA expression by qPCR demonstrated no TIMP-1 regulation of Fn mRNA expression in primary TIMP-1KO astrocyte cultures. (H) Validation of immunodepletion of Fn from ACM from primary WT and TIMP-1KO astrocyte cultures. (I) Effect of immunodepletion of Fn from ACM on OL differentiation in vitro in TIMP-1KO and Wt (control) treated cultures. Data in F–I were analyzed by ANOVA with either Bonferroni or Fisher’s least significant difference (LSD) post-hoc tests where appropriate *P < 0.05, **P < 0.01 as indicated. n = 3 biological replicates representing 3 to 4 averaged technical replicates/biological replicate.

Article Snippet: Anti- Fibronectin antibody (Biotin) (ab6584) (Abcam; rabbit, 1:5,000), Anti- Fibronectin antibody (ab23750) (Abcam; rabbit, 1:100- immunodepletion), Anti- Fibronectin antibody (ab2033) (Abcam; rabbit, 1:200), Anti- Timp1 antibody (AF980) (R&D Systems; goat, 1:125), Anti- MBP antibody (12) (Millipore; rat monoclonal, 1:500), Mouse anti- A2B5 (Invitrogen, Carlsbad, CA; 3 μg/mL), DAPI (Invitrogen, 1:1,000), Mouse anti- GFAP (Millipore, 1:400), Beta- actin (Sigma Aldrich, St. Louis, MO; mouse, 1:10,000).

Techniques: Liquid Chromatography, Mass Spectrometry, Western Blot, Expressing, Biomarker Discovery, Immunodepletion, In Vitro, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses.

doi: 10.1073/pnas.2306816121

Figure Lengend Snippet: Fig. 5. Characterization of Fn and TIMP-1 expression in primary human astrocytes and identification of Ana in human MS proteomic databases. (A) Schematic representation of two modes of analysis of human astrocytes using direct ex vivo, (e.g., scRNAseq) which has indicated no expression or no changes in TIMP-1 expression in astrocytes in the MS brain, and, as used in the present study, in vitro cultures from patient donors. (B) Relative FN1, TIMP1 and ratio FN1 to TIMP1 mRNA expression in control human (ctrl) vs. MS astrocytes as analyzed by qRT-PCR. (C) FN, TIMP1, and ratio FN to TIMP1 protein expression in ctrl human vs. MS astrocytes as analyzed by western blot. (D) Expression of Fn protein in ctrl human and MS astrocytes after TIMP-1 treatment (10 ng/mL human rTIMP-1 for 48 h). The dotted line indicates no treatment (untreated), which was set to 1. * indicates significant difference from untreated control (P < 0.05), one sample t test); ** indicates a significant difference between the two groups (P < 0.01, unpaired student t test). Non-demented control (blue shades 3 to 6) and MS (orange shades, 3 to 6) donors are consistently color-coded in B–D. (E) Proteomic analysis reveals Fn and Ana in the CSF of MS patients. “Low” indicates low cytokine levels in the CSF, while “High” indicates high cytokine levels in the CSF. A and B represent technical replicates.

Article Snippet: Anti- Fibronectin antibody (Biotin) (ab6584) (Abcam; rabbit, 1:5,000), Anti- Fibronectin antibody (ab23750) (Abcam; rabbit, 1:100- immunodepletion), Anti- Fibronectin antibody (ab2033) (Abcam; rabbit, 1:200), Anti- Timp1 antibody (AF980) (R&D Systems; goat, 1:125), Anti- MBP antibody (12) (Millipore; rat monoclonal, 1:500), Mouse anti- A2B5 (Invitrogen, Carlsbad, CA; 3 μg/mL), DAPI (Invitrogen, 1:1,000), Mouse anti- GFAP (Millipore, 1:400), Beta- actin (Sigma Aldrich, St. Louis, MO; mouse, 1:10,000).

Techniques: Expressing, Ex Vivo, In Vitro, Control, Quantitative RT-PCR, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses.

doi: 10.1073/pnas.2306816121

Figure Lengend Snippet: Fig. 6. Proposed model for astrocytic TIMP-1 regulation of OPC differentiation by Ana. Astrocytes when stimulated express and release TIMP-1, which under non-disease conditions would limit the production of Ana from fibronectin. In the absence of astrocytic TIMP-1, like which can occur under chronic inflammatory conditions, the elevated production of Ana impairs OPCs’ differentiation while also blocking the ability of FTY720 to promote differentiation. Together, these factors culminate in reduced remyelination of axons contributing to chronic demyelinating plaques in the CNS of MS patients.

Article Snippet: Anti- Fibronectin antibody (Biotin) (ab6584) (Abcam; rabbit, 1:5,000), Anti- Fibronectin antibody (ab23750) (Abcam; rabbit, 1:100- immunodepletion), Anti- Fibronectin antibody (ab2033) (Abcam; rabbit, 1:200), Anti- Timp1 antibody (AF980) (R&D Systems; goat, 1:125), Anti- MBP antibody (12) (Millipore; rat monoclonal, 1:500), Mouse anti- A2B5 (Invitrogen, Carlsbad, CA; 3 μg/mL), DAPI (Invitrogen, 1:1,000), Mouse anti- GFAP (Millipore, 1:400), Beta- actin (Sigma Aldrich, St. Louis, MO; mouse, 1:10,000).

Techniques: Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: Elevated Serum Gastrin Is Associated with Melanoma Progression: Putative Role in Increased Migration and Invasion of Melanoma Cells

doi: 10.3390/ijms242316851

Figure Lengend Snippet: Conditioned media from G17-stimulated melanoma cells exhibit upregulation of MMP-2 and downregulation of TIMP-3 expression. Representative demonstration of the confirmation of proteomic data using Western blot analysis, with prosaposin serving as a benchmark for gastrin responsiveness ( A ). Mean and standard deviation (±SD) of the densitometric analysis results from three independent experiments ( B ). Statistical difference (* p < 0.05) between treated and untreated control groups (considered as 100%) are indicated as follows: prosaposin, TIMP-3 and TIMP-1 levels in G361 cells and MMP2 expressions in the SK-MEL-2 cell line.

Article Snippet: Antibodies against MMP-2, TIMP-3 (R&D Systems Abingdon, UK; AF902 and MAB973, respectively), TIMP-1, TIMP-2, prosaposin, and MMP-1 (R&D Systems, Abingdon, UK; AF980, AF971, AF8520, and MAB901, respectively) were utilized.

Techniques: Expressing, Western Blot, Standard Deviation, Control