Cells cultured in a 10-centimeter dish (5 × 104 cells/plate) were treated with different concentrations of UA for 48 h; then, cell lysates were processed for Western blot analysis. Primary antibodies were used in 1 : 1000 dilutions for anti-phospho-AKT (AF0016, Affinity), anti-AKT (AF6261, Affinity), anti-phospho-mTOR (5536 s, Cell Signaling Technology), anti-mTOR (2983 s, Cell Signaling Technology), anti-phospho-p70S6K (AF3228, Affinity), anti-p70S6K (AF6226, Affinity), and anti-phospho-4E-BP1 (2855 s, Cell Signaling Technology). Additional primary antibodies used were anti-4E-BP1 (1 : 500, 60246-1-Ig, Proteintech), anti-p62 (1 : 750, AP6006, Bioworld, MN, USA), anti-LC3A/B (1 : 600, 14600-1-AP, Proteintech), and anti-β-actin (1 : 20000, AF7018, Affinity).
Af6226
Manufactured by Affinity Biosciences
Sourced in United States
AF6226 is a laboratory instrument designed for the quantitative detection and analysis of proteins. It utilizes an electrochemical detection method to measure the levels of specific proteins in samples.
Automatically generated - may contain errors
Lab products found in correlation
Af3228, by Affinity Biosciences (3 mentions)
Af6261, by Affinity Biosciences (2 mentions)
Af7018, by Affinity Biosciences (2 mentions)
Af6308, by Affinity Biosciences (2 mentions)
Af0016, by Affinity Biosciences (1 mentions)
Enhanced chemiluminescencent detection kit, by Yeasen (1 mentions)
S0001, by Affinity Biosciences (1 mentions)
Af1009, by Affinity Biosciences (1 mentions)
Af3308, by Affinity Biosciences (1 mentions)
Ecl detection reagent, by Yeasen (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Af0639, by Affinity Biosciences (1 mentions)
Af6423, by Affinity Biosciences (1 mentions)
Af3423, by Affinity Biosciences (1 mentions)
Af6241, by Affinity Biosciences (1 mentions)
Af5006, by Affinity Biosciences (1 mentions)
Af5301, by Affinity Biosciences (1 mentions)
Beyoecl plus, by Beyotime (1 mentions)
Ripa lysate, by Beyotime (1 mentions)
4 protocols using af6226
1
Protein Expression Analysis of UA Treatment
2
Placental Protein Expression Analysis
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Placental and trophoblastic protein expressions for LAT1, VDR, p70S6K1, and phospho-p70S6K1 were examined by western blot. Antibody against human LAT1 (sc-134994) was purchased from Santa Cruz Biotechnology (CA, USA), antibodies against human VDR (AF6159), p70S6K1 (AF6226), and phospho-p70S6K1 (AF3228) were all obtained from Affinity Biosciences (Jiangsu, China). An aliquot of 10 μg of tissue of cellular protein was subject to electrophoresis. The bound antibody was visualized with an enhanced chemiluminescencent detection kit (Yeasen, Shanghai, China). The bands for LAT1, VDR, and p70S6K1 were detected at 55KD, 48KD and 70KD, respectively. The band density was analyzed by ImageJ software (National Institutes of Health, USA). β-actin expression was determined and used to normalize relative protein expression in each sample.
3
Protein Expression Analysis in NSCLC Cells
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Briefly, PMSF and RIPA lysis buffer (Beyotime) were employed to extract the NSCLC cell proteins. To detect the concentration of cell proteins, Pierce BCA Protein assay kit was conducted. Cell proteins were isolated by 10% SDS‐PAGE before transferring to PVDF membranes. Using 5% skim milk, we blocked PVDF membranes at room temperature for 1 h. Later, with primary antibodies, the membranes were incubated overnight at 4°C, including UCP2 (1:1000, #DF8626, Affinity, USA), mTOR (1:500, #AF6308, Affinity), p‐mTOR(Ser2448) (1:500, #AF3308, Affinity), S6K (1:500, #AF6226, Affinity), p‐S6K(Thr389) (1:500, #AF3228, Affinity), 4E‐BP (1:500, #AF6432, Affinity), p‐4E‐BP(Thr70) (1:500, #AF2308, Affinity), HIF‐1α (1:500, #AF1009, Affinity) and α‐Tubulin (1:1000, #AF4651, Affinity). Next day, with HRP‐linked secondary antibody (1:3000, #S0001, Affinity), the membranes were washed before incubation at room temperature for 2 h. Finally, the membranes were visualized by ECL Detection Reagent (Yeasen, China), and ImageJ software was used to quantify the relative grayscale value.
4
Protein Extraction from Intestinal Tissue
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Protein extraction from intestinal tissue was performed using RIPA lysate (Beyotime Biotechnology, Shanghai, China). The extraction process for intestinal nucleoproteins was based on methods employed in a previous study (Shi et al., 2022a (link)). Experimental techniques for gel electrophoresis, membrane transfer, and antibody incubation were consistent with those detailed in the earlier study (Shi et al., 2022a (link)). Primary antibodies used included β-actin, p-AMPK, AMPK, PPARα, TOR, NF-κB p65, Nrf2, p70S6K, PI3K, and AKT (rabbit, 1:1000), with item no. AF7018, AF3423, AF6423, AF5301, AF6308, AF5006, AF0639, AF6226, AF6241, and AF6261, respectively (Affinity Biosciences, Jiangsu, China). IgG (rabbit, 1:2000, item no. S0001, Affinity Biosciences, Jiangsu, China) served as the secondary antibodies in the experiment. BeyoECL plus (Beyotime Biotechnology, Shanghai, China) was employed for protein band detection, and visualization was accomplished using the Genesys imaging system (Alcatel, Nanterre, France). Subsequently, Image J software (v1.54, Bethesda, MD, USA) was utilized to assess the gray value.
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