Fstl1

Manufactured by R&D Systems

Sourced in China, United Kingdom

FSTL1 is a laboratory reagent produced by R&D Systems. It is a recombinant human Follistatin-like 1 (FSTL1) protein. FSTL1 is an extracellular glycoprotein that belongs to the follistatin family.

Automatically generated - may contain errors

Lab products found in correlation

Stem cell factor (scf), by BioLegend (2 mentions) Bone morphogenetic protein 4 (bmp4), by BioLegend (2 mentions) Midkine, by OriGene (2 mentions) Betacellulin, by BioLegend (2 mentions) Gdf15, by MyBioSource (2 mentions) Mbs205834, by BioLegend (2 mentions) Ephrin a1, by BioLegend (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Collagen 1, by Cell Signaling Technology (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Easysep direct human neutrophil isolation kit, by STEMCELL (1 mentions) Interferon γ ifnγ, by R&D Systems (1 mentions) Interleukin 4 il 4, by R&D Systems (1 mentions) Interleukin 13 il 13, by R&D Systems (1 mentions) Leukotriene c4 ltc4, by Cayman Chemical (1 mentions) E selectin, by Thermo Fisher Scientific (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Gm csf, by R&D Systems (1 mentions) Lipopolysaccharide lps, by Enzo Life Sciences (1 mentions)

9 protocols using fstl1

Neutrophil Isolation and Stimulation

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Neutrophils were isolated from peripheral blood using an EasySep direct human neutrophil isolation kit (Stemcell, Vancouver, BC). Cells were then counted and cultured at 10⁷ cells/mL in RPMI supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin. Neutrophil cultures were treated with GM-CSF (1–25 ng/mL, R&D Systems, Minneapolis, MN), FSTL1 (100–1000 ng/mL, R&D Systems), Interferon-γ (IFNγ) (10 ng/mL, R&D Systems), lipopolysaccharide (LPS) (1 μg/mL, Enzo Life Sciences, Farmingdale, NY), Interleukin 4 (IL-4) (20 ng/mL, R&D Systems), Interleukin 13 (IL-13) (20 ng/mL, R&D Systems), Interleukin 25 (IL-25) (10–100ng/mL, R&D Systems), Interleukin 33 (IL-33) (10–100 ng/mL, R&D Systems), thymic stromal lymphopoietin (TSLP) (10–100 ng/mL, R&D Systems) or Leukotriene C₄ (LTC₄) (10⁻⁶ –10⁻⁷ Cayman Chemical, Ann Arbor, MI) for 20 hours, cell culture supernatants were collected for protein analysis and cell lysates were collected to isolate RNA. Plates were coated with E-selectin, ICAM (50ng/well, Peprotech, Rocky Hill, NJ) or bovine serum albumin (BSA) (50ng/well, Sigma Aldrich) in pH 8 tris buffered saline (TBS) overnight at 4 degrees. Neutrophils were seeded into the wells and the culture supernatants were harvested at 20 hours.

Pothoven K.L., Norton J.E., Suh L.A., Carter R.G., Harris K.E., Biyasheva A., Welch K., Shintani-Smith S., Conley D.B., Liu M.C., Kato A., Avila P.C., Hamid Q., Grammer LC I.I.I., Peters A.T., Kern R.C., Tan B.K, & Schleimer R.P. (2016). Neutrophils are a major source of the epithelial barrier disrupting cytokine Oncostatin M in mucosal airways disease. The Journal of allergy and clinical immunology, 139(6), 1966-1978.e9.

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Mature DCs Stimulation Assay

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Mature DCs were collected and separated into the following 4 groups: (1) NPC patients (mixed cells + FSTL1), (2) control group (NPC patient mixed cells), (3) healthy control sample 1 (mixed cells + FSTL1), and (4) healthy control samples 2 (mixed cells). Mixed cells refer to mature DCs and lymphocytes. FSTL1 (R&D Systems Inc., Minneapolis, MN) was diluted to a final concentration was 40 ng/mL. After 24 h, the DCs were washed twice with PBS and then stimulated with a mixture of EBV (LMP2) (JPT Peptide Technologies, Berlin, Germany) and a peptide fragment with EBV antigenic characteristics and cocultured with corresponding lymphocytes from the same NPC patient or healthy donor.

Wang H., Wu S., Huang S., Yin S., Zou G., Huang K., Zhang Z., Tang A, & Wen W. (2016). Follistatin‐like protein 1 contributes to dendritic cell and T‐lymphocyte activation in nasopharyngeal carcinoma patients by altering nuclear factor κb and Jun N‐terminal kinase expression. Cell Biochemistry and Function, 34(8), 554-562.

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Protein Expression and Signaling Pathway Analysis

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Cells were lysed in the RIPA buffer with protease inhibitor and protein phosphatase inhibitor. Protein concentration was determined with BCA Protein assay kit (Thermo Scientific, Rockford, IL, USA), the amount of protein sample loading was 40–60 μg per lane. The primary antibodies were as follows: FSTL1 (1:2000, AF1738 R&D Systems), PPARγ (1:1000, #2443 Cell Signaling Technology), p-PPARγ (phospho-Ser112) (1:1000, LS-C416660 LifeSpan BioSciences), ERK1/2 (1:1000, #4695 Cell Signaling Technology), p-ERK1/2 (1:1000, #4370 Cell Signaling Technology), p-FAK (phospho Y397) (1:1000, ab81298), p-FAK (phospho Y576/577) (1:1000, #3281 Cell Signaling Technology), p-FAK (phospho Y925) (1:1000, #3284 Cell Signaling Technology), FAK (1:1000, #3285 Cell Signaling Technology), Flag (1:1000, #14793 Cell Signaling Technology), HA (1:1000, sc-7392 SANTA CRUZ), ITGB1 (1:1000, 26918-1-AP Proteintech), COLLAGEN1 (1:1000, ab270993), GAPDH (1:3000, #5174 Cell Signaling Technology).

Fang D., Shi X., Jia X., Yang C., Wang L., Du B., Lu T., Shan L, & Gao Y. (2021). Ups and downs: The PPARγ/p-PPARγ seesaw of follistatin-like 1 and integrin receptor signaling in adipogenesis. Molecular Metabolism, 55, 101400.

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Recombinant Proteins Affect NE Cell Viability

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20,000 NE cells were seeded per well of a 96-well plate in RPMI media with 2% BGS in the presence of the recombinant proteins. Cell viability was assayed after 72 hours by the AlamarBlue® assay. The following recombinant proteins were used: Midkine (OriGene TP723299, 50ng/ml), Betacellulin (BioLegend 551302, 5ng/ml), Gdf15 (MyBioSource MBS205834, 25ng/ml), Bmp4 (BioLegend 595301, 50ng/ml), Ephrin A1 (BioLegend 755002, 50ng/ml), SCF (BioLegend 579702, 50ng/ml), Fstl1 (R&D Systems 1738-FN-050, 200ng/ml).

Lim J.S., Ibaseta A., Fischer M.M., Cancilla B., O’Young G., Cristea S., Luca V.C., Yang D., Jahchan N.S., Hamard C., Antoine M., Wislez M., Kong C., Cain J., Liu Y.W., Kapoun A.M., Garcia K.C., Hoey T., Murriel C.L, & Sage J. (2017). Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer. Nature, 545(7654), 360-364.

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Fstl1 Protein Quantification via Western Blot

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Tissue samples (50 mg) were solubilized in Ripa Buffer (89900, Thermo Fisher, Waltham, MA, USA) with addition of EASYpack protease inhibitors (5892970001, Roche). Protein concentration in supernatants after 14,000 rpm centrifugation was determined using the BCA method. 25 mkg of protein were separated on 8% SDS-PAGE with subsequent blotting on nitrocellulose membrane (BIO-RAD) using Trans-Blot^® Turbo™ Transfer System (Bio-Rad) with subsequent immersion in 5% skimmed milk in TBST buffer.
Primary ab: 1:1000-FSTL1, (BAF1738, R&D System); GAPDH (32233, Santa-Cruz). Secondary ab: 1:10000-anti-Mouse IgG (H+L) Secondary Antibody (115-005-003, Jackson ImmunoResearch), anti-Rabbit IgG (H+L) Secondary Antibody (111-035-144, Jackson ImmunoResearch). Signal was revealed by the ECL method following the HRP reaction.

Andreichenko I.N., Tsitrina A.A., Fokin A.V., Gabdulkhakova A.I., Maltsev D.I., Perelman G.S., Bulgakova E.V., Kulikov A.M., Mikaelyan A.S, & Kotelevtsev Y.V. (2019). 4-methylumbelliferone Prevents Liver Fibrosis by Affecting Hyaluronan Deposition, FSTL1 Expression and Cell Localization. International Journal of Molecular Sciences, 20(24), 6301.

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Western Blot Analysis of Protein Signaling

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Protein lysates were processed for western blot analysis following the standard protocol [21 (link), 22 (link)]. The following primary antibodies were used to recognize the proteins: p-Akt (Ser473), Akt, p-GSK-3β (Ser9), GSK-3β (Cell Signaling Technology), Fstl1 (R&D), Vimentin (Abcam), α-SMA, and GAPDH (Santa Cruz). Immunoreactivity was detected by routine enzymatic chemiluminescence.

Shen H., Cui G., Li Y., Ye W., Sun Y., Zhang Z., Li J., Xu G., Zeng X., Zhang Y., Zhang W., Huang Z., Chen W, & Shen Z. (2019). Follistatin-like 1 protects mesenchymal stem cells from hypoxic damage and enhances their therapeutic efficacy in a mouse myocardial infarction model. Stem Cell Research & Therapy, 10, 17.

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Immunoblot Analysis of Fibrotic Markers

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Immunoblot analysis was performed as previously described [53 (link), 54 (link)] with use of the following antibodies: TGF-β1, collagen I, PDGFR-β, α-SMA, Smad3, p-Smad3 (Cell Signaling Technologies), Fstl1 (R&D Systems), IL-6, HSP47 (Santa Cruz Biotechnology), FSP1 (Sigma-Aldrich), FN1 (Sigma-Aldrich), and HRP-labeled goat anti-mouse IgG or goat anti-rabbit IgG antibody peroxidase-conjugated (ZSGB-BIO, Beijing, China). GAPDH (Hangzhou Goodhere Biotechnology, Hangzhou, China) was used as an internal control for normalization of protein expression.

Liu B., Bing Q., Li S., Han B., Lu J., Baiyun R., Zhang X., Lv Y., Wu H, & Zhang Z. (2019). Role of A2B adenosine receptor-dependent adenosine signaling in multi-walled carbon nanotube-triggered lung fibrosis in mice. Journal of Nanobiotechnology, 17, 45.

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Immunohistochemical Analysis of Breast Cancer Metastasis

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Human breast cancer samples were obtained from the Cancer Hospital of Huanxing Chaoyang District Beijing and were fixed in 4% paraformaldehyde (PFA) for 24 to 48 hours. Mouse metastatic lung tissue was dissected and fixed in PFA for 48 hours, and then stored in 70% alcohol. All samples were embedded in paraffin, and 5 µm sections were obtained using a Paraffin slicer (RM2235; Leica, Heidelberg, Germany). For hematoxylin and eosin (H&E) staining, lung tissue was stained with H&E. For immunohistochemistry, sections were deparaffinized and rehydrated, then incubated with primary antibodies (FSTL1, AF1694 and AF1738; R&D Systems) at 4℃ overnight, followed by a horseradish peroxidase secondary antibody (donkey anti-goat IgG H&L, ab97110; Abcam, Cambridge, UK) at 37℃ for 1 hour. Samples were developed using a diaminobenzidine solution (ZsBio, Beijing, China) for 2 minutes at room temperature. Sections were then dehydrated and affixed to coverslips.

Zhang Y., Xu X., Yang Y., Ma J., Wang L., Meng X., Chen B., Qin L., Lu T, & Gao Y. (2018). Deficiency of Follistatin-Like Protein 1 Accelerates the Growth of Breast Cancer Cells at Lung Metastatic Sites. Journal of Breast Cancer, 21(3), 267-276.

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Recombinant Proteins Affect NE Cell Viability

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