H460 cells were seeded in 96 well plates (5000 cells/well) in 10% serum RPMI. The next day, the cells were transfected with siRNA directed against RSK1/2/3 (siGenome, Dharmacon) using DharmaFECT 1 transfection reagent (Dharmacon). After 48 hours, the cells were starved in serum free RPMI for 6 hours prior to treatment with TNFα (0–1000 ng/ml). Cell Viability was measured after 24 hours of TNFα treatment using the CellTitre Blue assay (Promega). To verify siRNA knockdown of RSK1, 2 and 3 protein, whole cell lysates were harvested at 48 hours post transfection, run on SDS PAGE gels, transferred to nitrocellulose membrane and probed using isoform specific antibodies: RSK1 (#9333), RSK2 (#9340) and RSK3 (#9343, all from Cell Signaling). Secondary antibodies used were anti-rabbit-HRP and anti-mouse-HRP (GE healthcare). Blots were stripped and re-probed using a β-actin mouse polyclonal antibody (Santa Cruz) as a loading control.
β actin mouse polyclonal antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The β-actin mouse polyclonal antibody is a research-use laboratory reagent designed to detect the β-actin protein, a ubiquitous cytoskeletal protein found in eukaryotic cells. This antibody can be used to identify and quantify β-actin expression levels in various biological samples through techniques such as Western blotting, immunohistochemistry, and immunocytochemistry.
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Lab products found in correlation
Celltitre blue assay, by Promega (1 mentions)
Dharmafect 1 transfection reagent, by Horizon Discovery (1 mentions)
Total protein kit, by Nanjing Jiancheng (1 mentions)
Hif 1α rabbit polyclonal antibody, by Abcam (1 mentions)
Iκbα rabbit polyclonal antibody, by Abcam (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Mda kit, by Nanjing Jiancheng (1 mentions)
Sod kit, by Nanjing Jiancheng (1 mentions)
Ultraviolet spectrophotometer, by Hitachi (1 mentions)
2 protocols using β actin mouse polyclonal antibody
1
RSK Knockdown Effects on TNFα Response
2
Hypobaric Stress Response Evaluation
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The main instruments used in this study were hypobaric chamber (Guizhou Fenglei Aviation Ordnance Co., Ltd, China); fluorescence microscope (Nikon, Japan); ultraviolet spectrophotometer (HITACHI, Japan). The main regents used in this study were Vitamin E (Zhenjiang Medicine, China), MDA kit (Nanjing Jiancheng, China), SOD kit (Nanjing Jiancheng, China), Total protein kit (Nanjing Jiancheng, China), 4% formaldehyde (Beijing Dingguo Changsheng Biotechnology Co., Ltd.). Rat IL-2, IL-4, TNF-α, INF-γ, S-IgA, EPO and DAO ELISA kits (R&D systems, USA); TLR4 and NF-κB p65 rabbit polyclonal antibody, occludin rabbit polyclonal antibody, and β-actin mouse polyclonal antibody (Santa Cruz, USA); HIF-1α rabbit polyclonal antibody (EPI); HIF-2α rabbit polyclonal antibody (Abcam, USA); IκBα rabbit polyclonal antibody (Abcam, USA) and P-IκBα rabbit polyclonal antibody (CST, USA).
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