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Anti tlr9

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Anti-TLR9 is a laboratory reagent used for the detection and analysis of Toll-like receptor 9 (TLR9) in various biological samples. TLR9 is an important component of the innate immune system and plays a role in the recognition of pathogen-associated molecular patterns. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and flow cytometry to study the expression and localization of TLR9 in cells and tissues.

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Lab products found in correlation

Anti gapdh, by Santa Cruz Biotechnology (3 mentions) Anti arrdc1, by Abcam (2 mentions) Anti mda5, by Abcam (2 mentions) Anti atg12, by Santa Cruz Biotechnology (2 mentions) Anti pink1, by Santa Cruz Biotechnology (2 mentions) Anti parkin mouse monoclonal clone prk8, by Santa Cruz Biotechnology (2 mentions) Anti miro, by Santa Cruz Biotechnology (2 mentions) Anti cd9 mm2 57, by Merck Group (2 mentions) Anti cd63 mem 259, by Thermo Fisher Scientific (2 mentions) Anti human mfge8, by Abnova (2 mentions) Anti dicer d11, by Santa Cruz Biotechnology (2 mentions) Anti tlr 7, by Cell Signaling Technology (2 mentions) Anti nf κb p50 alexa fluor 488 e 10, by Santa Cruz Biotechnology (2 mentions) Tcs nt upright confocal microscope, by Leica camera (2 mentions) Anti lc3, by Cell Signaling Technology (2 mentions) Beta actin ac 15, by Merck Group (2 mentions) Anti lc3, by Abcam (2 mentions) Anti tlr4, by Abcam (1 mentions) Facsaria 3, by BD (1 mentions) Anti tlr2, by Abcam (1 mentions)

9 protocols using anti tlr9

1

Toll-Like Receptor Expression in AOSD

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Twenty patients with AOSD and 15 HC subjects were used for fluorescence-activated cell sorting (FACS) analysis. PBMCs were isolated using CPT™ Mononuclear Cell preparation tube-BD Vacutainer® (BD Biosciences, Franklin Lakes, NJ, USA). For anti-TLR7 and TLR9 intracellular antibody reactions, cells were pre-treated with Tween 20. Cells (1 × 106) in each tube were incubated with FACS blocking buffer (3% BSA in PBS) at 4 °C for 1 h. After blocking, fluorescein isothiocyanate (FITC)-labelled anti-TLR1 (Abcam, Cambridge, MA, USA), anti-TLR2 (Abcam), anti-TLR7 (Thermo Fisher Scientific Inc., Waltham, MA, USA), and anti-TLR9 (Abcam) and phycoerythrin-labelled anti-TLR4 (Abcam) were added and incubated for 1 h at 4 °C. The same colour-labelled antibodies were applied separately to different tubes for isotype control. The stained cells were then washed with FACS buffer and analysed for 10,000 cells using a flow cytometer (FACSAria III; Becton, Dickinson and Company, San Jose, CA, USA). The FACS data were based on specific markers and were used to analyse the gated populations. The density was plotted using FlowJo V.10 software (Becton, Dickinson and Company, Ashland, OR, USA).
Han J.H., Ahn M.H., Jung J.Y., Kim J.W., Suh C.H., Kwon J.E., Yim H, & Kim H.A. (2022). Elevated expression of TLR2 and its correlation with disease activity and clinical manifestations in adult-onset Still’s disease. Scientific Reports, 12, 10240.
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2

Western Blotting for TLR4 and TLR9 Expression

Cited 2 times
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Cell pellets were processed for western blotting as previously described (Redfern et al., 2011 (link)). The membranes were probed with anti-TLR4 (0.2 μg/ml, Santa Cruz Biotechnology) or anti-TLR9 (1 μg/ml, AbCam; Cambridge, MA) antibodies, then incubated with horseradish peroxidase-conjugated secondary antibody, and visualized with ECL Plus (GE Healthcare; Piscataway, NJ). The membranes were stripped and reprobed using a GAPDH antibody as previously described (Giddabasappa et al., 2011 (link)). Densitometry measurements were obtained from non-saturated blots and the pixel intensity was normalized to GAPDH. Data are representative of a minimum of three experiments and were analyzed using an unpaired Student's t-test, with P ≤ 0.05 considered as statistically significant.
Redfern R.L., Barabino S., Baxter J., Lema C, & McDermott A.M. (2015). Dry Eye Modulates the Expression of Toll-Like Receptors on the Ocular Surface. Experimental eye research, 134, 80-89.
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3

Western Blot Analysis of Autophagy Markers

Cited 1 time
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Western blot analysis was conducted as previously described44 (link). The following primary (at a concentration of 1.5 μg ml−1) and secondary (0.5 ng ml−1) antibodies were used for western blotting: polyclonal anti-ARRDC1, anti-LC3, anti-MDA5, anti-TLR9 (Abcam), polyclonal anti-Atg-12 (Santa Cruz Biotechnology, SC-68884), polyclonal anti-Pink1 (Santa Cruz Biotechnology, SC-33796), anti-Parkin mouse monoclonal (clone PRK8, Santa Cruz Biotechnology), polyclonal anti-Miro (Santa Cruz Biotechnology, SC-292547), anti-CD9 (MM2/57, Millipore), anti-CD63 (MEM-259, ThermoFisher), anti-TSG101 (51/TSG101, BD Biosciences), anti-Human MFGE8 (Abnova), anti-Dicer (D11, Santa Cruz Biotechnology, used at a concentration of 2 μg ml−1), anti-Tubulin, anti-LC3 (rabbit polyclonal), anti-TLR-7 (Cell Signaling Technology), Beta actin (AC-15, Sigma-Aldrich) and anti-GAPDH (Santa Cruz Biotechnology, SC-25778). NF-κB nuclear translocation was characterized by staining cells with anti NF-κB-P50 Alexa Fluor 488 (E-10, Santa Cruz Biotechnology). Images were obtained using a Leica TCS NT upright confocal microscope.
Phinney D.G., Di Giuseppe M., Njah J., Sala E., Shiva S., St Croix C.M., Stolz D.B., Watkins S.C., Di Y.P., Leikauf G.D., Kolls J., Riches D.W., Deiuliis G., Kaminski N., Boregowda S.V., McKenna D.H, & Ortiz L.A. (2015). Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs. Nature Communications, 6, 8472.
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4

Immunomodulatory Antibodies and Cytokines

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The antibodies used in this study are listed as follows: anti-IL-12p40 (H306), Santa Cruz Biotechnology, Cat# SC-7926; GFAP, abcam, AB53554; GFAP (GA5), Cell Signaling Technology, Cat# 3670; anti-TLR9, Abcam, ab134368; anti-PARP (H250), Santa Cruz Biotechnology, Cat# SC-7150; anti-GAPDH, GeneTex, Cat# GTX100118. Primary antibodies used for neutralization include p40 (C17.8), or isotype control antibodies, BD Biosciences. The recombinant proteins used in this study to treat SHS-Y5Y cell lines: Murine IL-12 p70; Murine IL-12 p40 and Murine IL-12 p80, PeproTech.
Lai R.H., Chow Y.H., Chung N.H., Chen T.C., Shie F.S, & Juang J.L. (2022). Neurotropic EV71 causes encephalitis by engaging intracellular TLR9 to elicit neurotoxic IL12-p40-iNOS signaling. Cell Death & Disease, 13(4), 328.
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5

Protein Expression Analysis by Western Blot

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Western blotting was performed as described in our previous study 40 (link). Briefly, protein was isolated from lung tissues or cells, separated by 12% SDS-PAGE, and transferred to PVDF membranes. The membranes were then probed with the following primary antibodies: anti-GPX4, anti-TLR9, anti-MyD88, anti-p65, anti-METTL3 (Abcam) and anti-GAPDH (Santa Cruz Biotechnology). The antibodies were incubated overnight at 4 °C and further visualized using enhanced western blot chemiluminescence detection reagents (Amersham Biosciences Inc., Piscataway, NJ, USA).
Zhang H., Liu J., Zhou Y., Qu M., Wang Y., Guo K., Shen R., Sun Z., Cata J.P., Yang S., Chen W, & Miao C. (2022). Neutrophil extracellular traps mediate m6A modification and regulates sepsis-associated acute lung injury by activating ferroptosis in alveolar epithelial cells. International Journal of Biological Sciences, 18(8), 3337-3357.
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6

Western Blot Analysis of Autophagy and Inflammation Markers

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Western blot analysis was conducted as previously described 44 (link). The following primary (at a concentration of 1.5 μg/ml) and secondary (0.5 ng/ml) antibodies were used for Western Blotting: polyclonal anti-ARRDC1, anti-LC3, anti-MDA5, anti-TLR9 (Abcam), polyclonal anti-Atg-12 (Santa Cruz Biotechnology, SC-68884), polyclonal anti-Pink1 (Santa Cruz Biotechnology, SC-33796), anti-Parkin mouse monoclonal (clone PRK8, Santa Cruz Biotechnology), polyclonal anti-Miro (Santa Cruz Biotechnology, SC-292547), anti-CD9 (MM2/57, Millipore), anti-CD63 (MEM-259, ThermoFisher), anti-TSG101 (51/TSG101, BD Bioscciences), anti-Human MFGE8 (Abnova), anti-Dicer (D11, Santa Cruz Biotechnology, used at a concentration of 2 μg/ml), anti-Tubulin, anti-LC3 (rabbit polyclonal), anti-TLR-7 (Cell Signaling Technology), Beta actin (AC-15, Sigma Aldrich), anti-GAPDH (Santa Cruz Biotechnology, SC-25778). NFκB nuclear translocation was characterized by staining cells with anti NFκB -P50 Alexa Fluor 488 (E-10, Santa Cruz). Images were obtained using a Leica TCS NT upright confocal microscope.
Phinney D.G., Di Giuseppe M., Njah J., Sala E., Shiva S., St Croix C.M., Stolz D.B., Watkins S.C., Di Y.P., Leikauf G.D., Kolls J., Riches D.W., Deiuliis G., Kaminski N., Boregowda S.V., McKenna D.H, & Ortiz L.A. (2015). Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs. Nature Communications, 6, 8472.
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7

Aorta and Cell Protein Expression Analysis

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Lysates prepared from cells or aorta in RIPA buffer (Wako Pure Chemical Industries, Ltd, Osaka, Japan) containing a protease inhibitor cocktail (Roche, Basel, Switzerland) and a phosphatase inhibitor cocktail (Thermo Fisher Scientific) were separated on SDS–polyacrylamide gel electrophoresis gels. The following antibodies were used: anti‐TLR9, anti‐VCAM‐1 (Abcam, Cambridge, MA), anti‐p38 MAPK, anti‐phospho‐p38 MAPK (Cell Signaling, Danvers, MA), and anti‐β‐actin (Sigma, Fukushima, Japan). Levels of expression were quantified by densitometry (ImageQuant LAS 4000mini; GE Healthcare Life Sciences, Little Chalfont, England).
Fukuda D., Nishimoto S., Aini K., Tanaka A., Nishiguchi T., Kim‐Kaneyama J., Lei X., Masuda K., Naruto T., Tanaka K., Higashikuni Y., Hirata Y., Yagi S., Kusunose K., Yamada H., Soeki T., Imoto I., Akasaka T., Shimabukuro M, & Sata M. (2019). Toll‐Like Receptor 9 Plays a Pivotal Role in Angiotensin II–Induced Atherosclerosis. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 8(7), e010860.
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8

Immunofluorescence Analysis of Spinal Cord

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Mice were anesthetized with 10% chloral hydrate. Then they were transcardially perfused with saline and perfused with 4% formaldehyde. L4-L6 segment of the mice spinal cord was soaked in 4% formaldehyde for 4 h, and subsequently in 20 and 30% sucrose. The 30 μm-thick sections were incubated in Superblock Buffer (Thermo, 37580) for 1 h at room temperature, reacted with mouse anti-NeuN (Millipore) or anti-TLR9 (Abcam, ab37154) overnight at 4°C, and then reacted with Alexa-594-conjugated donkey anti-mouse IgG secondary antibodies (Invitrogen) for 2 h at room temperature. After three washes with PBST, the sections were attached using mounting media containing DAPI and were analyzed by confocal microscope (FV1000, Olympus). The mean gray value and the cell amount were analyzed by ImageJ. The Pearson’s R value was measured by the coloc 2 function of ImageJ.
Chen Y., Chen H., Li X.C., Mi W.L., Chu Y.X., Wang Y.Q, & Mao-Ying Q.L. (2022). Neuronal toll like receptor 9 contributes to complete Freund’s adjuvant-induced inflammatory pain in mice. Frontiers in Molecular Neuroscience, 15, 1008203.
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9

Western Blot Analysis of Fibroblast Proteins

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NHLFs were lysed in buffer (6.1 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 88.4 mmol/L NaCl, 36.6 mmol/L LiCl, 36.6 mmol/L SDS, 24.1 mmol/L sodium deoxycholate, and 1% Triton X-100) and complete protease inhibitor cocktail (Roche). Cell debris was removed by centrifugation, and supernatants were denatured at 100 C in Tris-glycine SDS sample buffer. Protein samples (10 mg) were subjected to electrophoresis on a 10% reducing SDS-PAGE gel (Bio-Rad) and blotted onto a nitrocellulose or polyvinylidene difluoride membrane (Bio-Rad Laboratories Incorporated). Membranes were blocked for 2 hours to overnight in Tris-buffered saline, 0.1% (v/v) Tween-20, and 5% (w/v) nonfat dried milk. Immunodetection was performed using anti-TLR9 (Abcam), anti-aSMA (Abcam), anti-PDGFRa (Cell Signaling), antieMMP-14 (Abcam), anti-fibronectin (Abcam), anti-CD44 (Santa Cruz Biotechnology, Dallas, TX), anti-cleaved caspase-3 (Cell Signaling, Danvers, MA), and antieglyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology). The protein bands were visualized using the ECL system (Denville Scientific, South Plainfield, NJ). All Western blot densitometry data are normalized for the loading control, glyceraldehyde-3phosphate dehydrogenase.
Kirillov V., Siler J.T., Ramadass M., Ge L., Davis J., Grant G., Nathan S.D., Jarai G, & Trujillo G. (2015). Sustained activation of toll-like receptor 9 induces an invasive phenotype in lung fibroblasts: possible implications in idiopathic pulmonary fibrosis. The American journal of pathology, 185(4).
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