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Nbp2 29328

Manufactured by Novus Biologicals
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NBP2-29328 is a recombinant protein product from Novus Biologicals. It is a purified protein produced in E. coli.

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Lab products found in correlation

Streptomycin, by Merck Group (2 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Harvard Bioscience (2 mentions) Penicillin, by Harvard Bioscience (2 mentions) Syk inhibitor r406, by InvivoGen (1 mentions) Mek inhibitor u0126, by Cell Signaling Technology (1 mentions) Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions) Uo126, by Cell Signaling Technology (1 mentions) Vas2870, by Merck Group (1 mentions) Ml171, by Merck Group (1 mentions) Diphenyleneiodonium chloride dpi, by Merck Group (1 mentions) Pkc inhibitor go 6976, by Merck Group (1 mentions) Nox2ds tat, by AnaSpec (1 mentions) Fugene hd transfection reagent, by Promega (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Gapdh, by Abcam (1 mentions) Anti rat alexa568, by Thermo Fisher Scientific (1 mentions) Anti fitc alexa 488, by Thermo Fisher Scientific (1 mentions)

6 protocols using nbp2 29328

1

Larval Infection Assay with Microalgae

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6 dpf larvae were infected with 1.0 x 105 cells ml-1 of B. minutum (SSB01, symbiont) or N. oculata for 24 hours. Infected larvae were washed with FASW to remove microalgae from water and 10 units ml-1 penicillin and 10 μg ml-1 streptomycin (P4333 Sigma-Aldrich) were added. Infected larvae were incubated for 24 hours at 26 °C under a 12L:12D cycle with either 50 μM of MyD88 inhibitor peptide or 50 μM control peptide (NBP2-29328, Novus Biologicals, Centennial, CO, USA). Larvae were fixed in 4% formaldehyde and infection efficiency was quantified as previously described.
Jacobovitz M.R., Rupp S., Voss P.A., Maegele I., Gornik S.G, & Guse A. (2021). Dinoflagellate symbionts escape vomocytosis by host cell immune suppression. Nature microbiology, 6(6), 769-782.
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2

Inhibition of SYK and MYD88 in BMDCs

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For SYK and MYD88 inhibition, BMDCs were pre-incubated with 1 µM SYK inhibitor (R406; InvivoGen, CA, USA) for 30 min or 100 µM MYD88 inhibitor peptide (NBP2–29328; Novus Biologicals, CO, USA) for 24 h prior to stimulation. The negative control of SYK inhibitor and MYD88 inhibitor peptide was DMSO and control peptide, respectively.
For TLR blockade, BMDCs were pre-incubated with 20 µg/ml of anti-mouse/human TLR2 mAb (Biolegend, CA, USA) for 2 h prior to stimulation. Rat IgG was used as the isotype control.
Nguyen T.N., Padungros P., Wongsrisupphakul P., Sa-Ard-Iam N., Mahanonda R., Matangkasombut O., Choo M.K, & Ritprajak P. (2018). Cell wall mannan of Candida krusei mediates dendritic cell apoptosis and orchestrates Th17 polarization via TLR-2/MyD88-dependent pathway. Scientific Reports, 8, 17123.
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3

Macrophage Isolation and Biglycan Stimulation

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Thioglycolate-elicited macrophages were isolated from peritoneal lavage of wild-type C57BL/6, Sphk1−/−, Sphk2−/−, Tlr2−/−, Tlr4−/−, Tlr2−/−/Tlr4-m and Trif-m mice and grown in RPMI 1640 (Life Technologies, Darmstadt, Germany) supplemented with 1% penicillin and streptomycin and 2% fetal bovine serum (Biochrom, Berlin, Germany). Cells were stimulated with 4 μg/mL (80 nM) purified human biglycan [7 (link)] in serum-free medium for the indicated time points. For purification of the native proteoglycan, containing two chondroitin/dermatan sulfate chains, the conditioned medium of human biglycan-overexpressed 293 HEK cells was collected, passed over a DEAE-Trisacryl-M (Pall) column and further purified through high performance liquid chromatography [8 (link)]. The purity of the bigycan was verified by silver staining after sodium dodecyl sulfate (SDS) gel electrophoresis. MyD88 inhibitory peptide NBP2-29328 (50 μM; Novus Biologicals, Wiesbaden, Germany) was applied 30 min prior to stimulation with biglycan. SphK1 inhibitor PF-543 (100 nM, Cayman Chemical, Hamburg, Germany), MEK inhibitor U0126 (10 μM, Cell Signaling, Frankfurt am Main, Germany), p38 MAPK inhibitor SB203580 (10 μM, Calbiochem, Darmstadt, Germany) and IKK inhibitor III (10 μM, Merck, Darmstadt, Germany) were applied 30 min prior to stimulation with biglycan.
Hsieh L.T., Nastase M.V., Roedig H., Zeng-Brouwers J., Poluzzi C., Schwalm S., Fork C., Tredup C., Brandes R.P., Wygrecka M., Huwiler A., Pfeilschifter J, & Schaefer L. (2017). Biglycan- and Sphingosine Kinase-1 Signaling Crosstalk Regulates the Synthesis of Macrophage Chemoattractants. International Journal of Molecular Sciences, 18(3), 595.
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4

Murine Macrophage Activation by Biglycan

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Murine thioglycolate-elicited macrophages were isolated from peritoneal lavage and grown in RPMI 1640 (Life Technologies, Germany) supplemented with 1% penicillin and streptomycin and 2% fetal bovine serum (Biochrom, Germany). Cells were stimulated with 4 μg/ml (80 nM) human biglycan in serum-free medium for indicated time periods. Phorbol 12-myristate 13-acetate (PMA) (100 nM; Sigma, Germany) served as positive control for p47phox translocation studies. For ROS inhibition diphenyleneiodonium chloride (DPI, 0.5 μM; Sigma, Germany), VAS2870 (5 μM; Sigma, Germany), ML-171 (10 nM; Merck, Germany), Nox2ds-tat (40 μM; AnaSpec, USA) and GKT137831 (200 μM) were applied to the macrophages 1 h prior to stimulation with biglycan. For immunofluorescence studies Akt inhibitor 124008 (1 μM; Calbiochem, Germany), p38 MAPK inhibitor SB203580 (10 μM; Calbiochem, Germany), MEK1/2 inhibitor UO126 (10 μM; Cell Signaling, Germany), PI3K inhibitor LY294002 (10 μM; Cell Signaling, Germany), Rac1 inhibitor (50 μM; Calbiochem, Germany), PKC inhibitor GO6976 (20 nM; Sigma, Germany) and MyD88 inhibitor peptide NBP2–29328 (50 μM; Novus Biologicals, Germany) were applied 1 h prior to stimulation with biglycan. For HSP70 inhibition phenylsulfonamide (PES, pifithrin-μ) (200 μM; Sigma, Germany) was applied to the cells in addition to biglycan.
Hsieh L.T., Frey H., Nastase M.V., Tredup C., Hoffmann A., Poluzzi C., Zeng-Brouwers J., Manon-Jensen T., Schröder K., Brandes R.P., Iozzo R.V, & Schaefer L. (2015). Bimodal role of NADPH oxidases in the regulation of biglycan-triggered IL-1β synthesis. Matrix biology : journal of the International Society for Matrix Biology, 49, 61-81.
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5

Comprehensive Immune Signaling Pathway Analysis

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Mouse or goat IgG directed against TLR9 or Cav-1 and rabbit IgG directed against TLR9, Cav-1, GAPDH, TLR4, IRAK-4, TRAF6, TRAF3, MyD88, IRF7, IRF3, p38MAPK and JNK were obtained from Abcam (Abcam, USA). Anti-Ly6G and anti-CD11b were from Abcam Plc (Cambridge, UK). Mouse TNF-α (RAB0477) and IL-6 (RAB0308) ELISA Kits were purchased from Sigma (Sigma-Aldrich China, Shanghai). MyD88 homodimerization inhibitor peptide (NBP2-29328) was purchased from Novus Biologicals (Novus, USA).
MPLA (L 5399) was from Sigma (Sigma, USA) TLR9-specific shRNA (sc-40270-SH), Cav-1 shRNA (sc-29241-SH) and transfection reagent (sc-108061) were bought from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-TLR9 for flow cytometry (560425, BD), Anti-Cav-1 for Flow Cytometry (PA1-064, Invitrogen), anti-MyD88 for flow cytometry (566354, BD), anti-IRF3 for flow cytometry (566347, BD), anti-TRAF3 for flow cytometry (PA5-29091, Invitrogen) and Fugene HD transfection reagents were obtained from Promega (Promega, USA). The full-length Cav-1 was ligated into pECFP-C1 and TLR9 was inserted into pEYFP-N1. Anti-mouse-IgG3-FITC was from Southern Biotech (Birmingham, USA); anti-human IgG-FITC, anti-rat Alexa568, and anti-rabbit Alexa555 were purchased from Thermo Fisher Scientific (Braunschweig, Germany); anti-FITC-Alexa488 was from Life Technologies (Darmstadt, Germany); and DAPI was from Sigma (Deisenhofen, Germany).
Yang Z., Wang L., Yu H., Wang R., Gou Y., Zhang M., Kang C., Liu T., Lan Y., Wang X., Liu J., Cooper M.A., Li X., Yue K., Yu Y., Wang L., Kim B.Y., Jiang W, & Sun W. (2019). Membrane TLR9 Positive Neutrophil Mediated MPLA Protects Against Fatal Bacterial Sepsis. Theranostics, 9(21), 6269-6283.
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6

Larval Infection Assay with Microalgae

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6 dpf larvae were infected with 1.0 x 105 cells ml-1 of B. minutum (SSB01, symbiont) or N. oculata for 24 hours. Infected larvae were washed with FASW to remove microalgae from water and 10 units ml-1 penicillin and 10 μg ml-1 streptomycin (P4333 Sigma-Aldrich) were added. Infected larvae were incubated for 24 hours at 26 °C under a 12L:12D cycle with either 50 μM of MyD88 inhibitor peptide or 50 μM control peptide (NBP2-29328, Novus Biologicals, Centennial, CO, USA). Larvae were fixed in 4% formaldehyde and infection efficiency was quantified as previously described.
Jacobovitz M.R., Rupp S., Voss P.A., Maegele I., Gornik S.G, & Guse A. (2021). Dinoflagellate symbionts escape vomocytosis by host cell immune suppression. Nature microbiology, 6(6), 769-782.
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