Af6294

Manufactured by Affinity Biosciences

Sourced in China, United States

AF6294 is an analytical instrument designed for the quantitative detection and analysis of specific target molecules or compounds in a sample. It utilizes sensitive detection methods to provide accurate and reliable measurements.

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Lab products found in correlation

4 protocols using af6294

Immunohistochemical Analysis of Inflammatory Factors in Colon

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Colon tissue was sealed with wax and sectioned into 4 μm thick slices. Sectioned samples were deparaffinized in xylene, rehydrated with gradient alcohol, and subjected to antigen retrieval. Colon sections were washed with PBS five times for 3 min each. Then, 0.5% (wt/vol) Triton X-100 and blocking serum were added successively and incubated for 10 min and 1.5 h, respectively. The tissue was incubated in primary antibody, rabbit TNF-α (1:200, AF7014, Affinity Biosciences Inc., China), rabbit IL1-β(1:200, AF5103, Affinity Biosciences Inc., China), rabbit IL-6 (1:200, DF6087, Affinity Biosciences Inc., China), rabbit JAK2 (1:200, AF6022, Affinity Biosciences Inc., China), rabbit STAT3 (1:200, AF6294, Affinity Biosciences Inc., China), rabbit F4/80(1:200, DF7762, Affinity Biosciences Inc., China), at 4 ℃ overnight. After being washed four times with PBS, the sections were incubated with the secondary antibody (ZF-0516, Alexa Fluor^® 594, ZSGB-Bio Inc., China) for 2 h at RT and protected from light. Images were obtained at 20× the original magnification. The relative area immunoreactivity was calculated with Image J software.

Lu Z., Xiong W., Xiao S., Lin Y., Yu K., Yue G., Liu Q., Li F, & Liang J. (2020). Huanglian Jiedu Decoction ameliorates DSS-induced colitis in mice via the JAK2/STAT3 signalling pathway. Chinese Medicine, 15, 45.

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Western Blot Analysis of Protein Expression

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Total protein lysates were extracted with cell lysis buffer (P0013, Beyotime) containing a protease inhibitor (ST506, Beyotime) and quantified with a BCA assay kit (P0011, Beyotime). Protein samples were loaded on SDS‒PAGE gels and transferred onto polyvinylidene difluoride membranes. After being blocked in 5% BSA for 1 h at room temperature, the membranes were hybridized with primary antibodies against JMJD1C (1:1000; A20153, ABclonal, Shanghai, China), HK2 (1:1000; A0994, ABclonal), PGK1 (1:1000; A12686, ABclonal), LDHA (1:1000; A0861, ABclonal), p-STAT3 (1:1000; AF3293, Affinity), STAT3 (1:1000; AF6294, Affinity), cyclin D1 (1:1000; A19038, ABclonal), CDK4 (1:1000; A11136, ABclonal) or β-actin (1:1000; sc-47778, Santa Cruz, Dallas, TX, USA) overnight at 4 °C, followed by goat anti-rabbit IgG (1:5000; A0208, Beyotime) or goat anti-mouse IgG (1:5000; A0216, Beyotime) secondary antibody incubation for 45 min at 37 °C. The protein signals were visualized by an enhanced chemiluminescence system (P0018, Beyotime) and quantified using Gel-Pro-Analyzer Software (WD-9413B, Beijing Liuyi Biotechnology Co., Ltd., Beijing, China).

Zhang C., Sun Y., Guo Y., Xu J, & Zhao H. (2023). JMJD1C promotes smooth muscle cell proliferation by activating glycolysis in pulmonary arterial hypertension. Cell Death Discovery, 9, 98.

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Ferroptosis Regulators and Cellular Signaling

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Bavachin was purchased from MCE (China), and pifithrin-α (PFT-α), deferoxamine (DFO), ferrostatin-1 (Fer-1), and liproxstatin-1 (Lip-1) were purchased from Topscience (China). Vitamin E was purchased from Beyotime (Shanghai, China). Rabbit polyclonal anti-transferrin receptor antibody (TFRC, AF5343, 1 : 1000), rabbit polyclonal anti-divalent metal transporter-1 antibody (DMT1, DF12740. 1 : 1000), rabbit polyclonal anti-ferritin light chain antibody (FTL, DF6604, 1 : 1000), rabbit polyclonal anti-ferritin heavy chain antibody (FTH, DF6278, 1 : 1000), rabbit polyclonal anti-SLC7A11 antibody (DF12509, 1 : 1000), rabbit polyclonal anti-P53 antibody (AF0879, 1 : 1000), rabbit polyclonal anti-STAT3 antibody (AF6294, 1 : 1000), rabbit polyclonal anti-p-STAT3 (705) antibody (AF3293, 1 : 1000), and rabbit polyclonal anti-glutathione peroxidase-4 antibody (GPX4, DF6701, 1 : 1000) were purchased from Affinity Bioscience (China). HRP goat anti-rabbit IgG was purchased from Earthox (USA).

Luo Y., Gao X., Zou L., Lei M., Feng J, & Hu Z. (2021). Bavachin Induces Ferroptosis through the STAT3/P53/SLC7A11 Axis in Osteosarcoma Cells. Oxidative Medicine and Cellular Longevity, 2021, 1783485.

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Cardiac TLR4, STAT3 Protein Analysis

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Toll-like receptor 4 (TLR4) (Affinity Biosciences Cat# AF7017, RRID: AB_2835322; Affinity Biosciences, Cincinnati, OH, USA), total signal transducer and activator of transcription 3 (T-STAT3) (Affinity Biosciences Cat# AF6294, RRID: AB_2835144) and its phosphorylated (Affinity Biosciences Cat# AF3293, RRID: AB_2810278) form in heart muscle were evaluated by Western blotting. Samples were homogenized in a lysis buffer, sonicated, and centrifuged. Protein concentration was determined using a bicinchoninic acid (BCA) protein quantitation assay. Protein (40 μg) were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, blocked with 5% bovine serum albumin (BSA) [in Tris-buffered saline with 0.1% Tween 20 (TBST)] for 2 h at room temperature and then incubated with primary antibodies overnight at 4°C. The membranes were then washed and incubated with secondary horseradish peroxidase-conjugated antibodies for 1 h at room temperature. After washing with TBST, bands were detected using the enhanced chemifluorescence reagent. The intra- and inter-assay variation was tested in GAPDH and β-actin, and western blotting efficiency of the two reference proteins were similar and constant. Band intensity was quantified by scanning densitometry using Image J software and normalized to GAPDH (Affinity Biosciences Cat# AF7021, RRID: AB_2839421).

Zhang Q.Q., Li G.Q., Zhong Y., Wang J., Wang A.N., Zhou X, & Mao X.M. (2020). Empagliflozin improves chronic hypercortisolism-induced abnormal myocardial structure and cardiac function in mice. Therapeutic Advances in Chronic Disease, 11, 2040622320974833.

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