Preprocessed samples were added to the RIPA lysate solution (P0013B; Beyotime, China), and the supernatants were collected after centrifugation. Samples were loaded onto sodium dodecyl sulfate-polyacrylamide gels (3250GR500; neoFROXX, Einhausen, Germany). Proteins were transferred to polyvinylidene difluoride membranes (WGPVDF22; Servicebio, China) after electrophoresis in transfer buffer for 5 min. The membranes were then incubated with primary antibodies (1:5000 dilution) targeting CD31 (AF6191, Affinity, USA), Vimentin (BF8006, Affinity), α-SMA (AF1032, Affinity), slug (350,136, ZENBIO, China), snail (AF6032, Affinity), twist (AF4009, Affinity), LC3II (3868S, CST, USA), p62 (AF5384, Affinity), beclin 1 (AF5128, Affinity), AMPK (AF6423, Affinity), p-AMPK (AF3423, Affinity), mTOR (AF6308, Affinity), and p-mTOR (AF3308, Affinity). Horseradish peroxidase-conjugated secondary antibodies at a 1:10,000 dilution were added after washing. The membranes were then viewed with an automatic darkroom exposure instrument (JS-M6P; P&Q, China) and varying luminous intensities were used for optimal exposure.
Af6308
Manufactured by Affinity Biosciences
Sourced in United States, China
AF6308 is a laboratory equipment product manufactured by Affinity Biosciences. It serves as a core functional device, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Af3308, by Affinity Biosciences (7 mentions)
Af0016, by Affinity Biosciences (3 mentions)
Af6261, by Affinity Biosciences (3 mentions)
Af6241, by Affinity Biosciences (3 mentions)
Af6423, by Affinity Biosciences (2 mentions)
Af3423, by Affinity Biosciences (2 mentions)
Af5128, by Affinity Biosciences (2 mentions)
Af5384, by Affinity Biosciences (2 mentions)
Af1009, by Affinity Biosciences (2 mentions)
Af6226, by Affinity Biosciences (2 mentions)
Af7018, by Affinity Biosciences (2 mentions)
Af1032, by Affinity Biosciences (1 mentions)
Ripa lysate solution, by Beyotime (1 mentions)
Af6191, by Affinity Biosciences (1 mentions)
Bf8006, by Affinity Biosciences (1 mentions)
Af6032, by Affinity Biosciences (1 mentions)
Af4009, by Affinity Biosciences (1 mentions)
Wgpvdf22, by Wuhan Servicebio Technology (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Bca kit, by Solarbio (1 mentions)
9 protocols using af6308
1
Western Blot Analysis of Cell Markers
2
Protein Expression Analysis of Key Signaling Pathways
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A RIPA lysate (Beyotime, China) was utilized to extricate total proteins, which were quantified by a BCA kit (Solarbio, China), followed by subjecting them to an SDS-PAGE gel and transferring them to PVDF membranes (GE Healthcare Life, USA). Afterwards, blocking blots were performed with 5% fat-free milk, and the process was followed by incubation with the primary antibodies against KRT17 (AF5480, Affinity, USA), E-cadherin (AF0131, Affinity), N-cadherin (AF4039, Affinity), β-catenin (AF6266, Affinity), AKT (AF6261, Affinity), p-AKT (AF0016, Affinity), mTOR (AF6308, Affinity), p-mTOR (AF3308, Affinity c-Myc (AF0358, Affinity), cyclin D1 (AF0931, Affinity), cyclin E (AF0144, Affinity), CDK2 (AF6237, Affinity), CDK4 (DF6102, Affinity), Snail (AF6032, Affinity), MMP7 (AF0218, Affinity), and GAPDH (AF7021, Affinity). Then, the membranes were encoded with a secondary antibody conjugated to HRP and finally subjected to measurement with an ECL kit (Pierce, Waltham, MA, USA).
3
Quantitative Western Blot Analysis
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Total proteins were extracted from cells and quantified with the BCA method, which were separated using the 12% SDS-PAGE, followed by transferred to the PVDF membrane. Following blocking, primary antibodies against RET (1:1000, AF6120, Affinity, USA), p-AKT (1:1000, AF0016, Affinity, USA), AKT (1:1000, AF6259, Affinity, USA), mTOR (1:1000, AF6308, Affinity, USA), p-mTOR (1:1000, AF3308, Affinity, USA), and GAPDH (1:10000, 1094-1-AP, Proteintech, USA) were added to be cultured for 12 h at 4 ℃, followed by incubation with the secondary antibody (1:6000, 7074, CST, USA) and incubated for 60 min. Lastly, the ECL solution was added for exposure and the expression level was quantified with the Image J software.
4
Western Blot Analysis of Gastric Tissue
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Total protein was extracted from the gastric tissue and SNU‐5 and AGS cell lines using conventional methods. Equal amounts of protein samples (15 μg each) and molecular weight markers (Thermo Fisher, USA) were separated using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and electrically transferred to a poly (vinylidene fluoride) membrane, which was then blocked in 5% skim milk for 2 h and incubated with the primary antibody overnight at 4°C. After incubation with secondary antibodies for 2 h at 25°C, protein bands were visualized using an enhanced chemiluminescence detection kit (Thermo Fisher, USA). They were also washed appropriately between each step using Tris‐buffered saline containing 0.1% Tween‐20 (TRIS‐buffered saline containing 0.1% Tween‐20). The following antibodies were used: KIRREL (BS‐6435R, Bioss, China), PI3K (AF6241, Affinity, China), P‐PI3K (AF3242, Affinity, China), AKT (AF6261, Affinity, China), P‐AKT (AF0832, Affinity, China), m‐TOR (AF6308, Affinity, China), P‐mTOR (AF3308, Affinity, China), HIF‐1α (AF1009, Affinity, China), VEGF (AF5131, Affinity, China) and GAPDH (TA‐08, ZSGB‐BIO, China).
5
Protein Expression Analysis of HUVECs
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HUVECs were cultured in 6-well plates for 24 h, then the total protein was extracted. Western blotting was performed to detect the protein expression levels of phosphorylated (p-ERK1/2, ERK1/2, p-mTOR and mTOR, with GAPDH as the reference gene. The procedure of western blotting followed the standard protocol. In brief, about 106 cells were taken and lysed and the total protein was obtained; a 10% SDS-PAGE gel was prepared; 30 µg total protein was loaded in each lane and gel electrophoresis was performed at 60 V for 30 min and then at 90 V for 1 h; the protein bands on the gel were transferred to a polyvinylidene difluoride (PVDF) membrane under a steady current of 200 mA for 120 min; the PVDF membrane was blocked with 5% skimmed milk powder for 2 h, then the membrane was incubated with appropriate antibodies for p-ERK1/2 (Affinity, AF1014), ERK1/2 (Affinity, AF0155), p-mTOR (Affinity, AF3308), mTOR (Affinity, AF6308) with a dilution ratio of 1:1,000 and GAPDH (ab37168; Abcam) as the reference with a dilution ratio of 1:2,000. For chemiluminescence detection, an ECL western blotting kit (Thermo Fisher Scientific, Inc.) was used to react with the interest proteins. Tanon Automatic Chemiluminescence Image Analysis System (5200, China) was used to scan the light bands 5 min after the reaction and the matching software accompanying the machine was used for analysis.
6
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.
7
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.
8
Western Blot Analysis of Apoptosis and Autophagy Markers
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Total protein of the left ventricular tissue of rats and H9c2 cells with LBAG treatment was extracted using lysis buffer containing PMSF on ice. The protein levels were estimated with a BCA method (Thermo Fisher Scientific). Equivalent protein (20 μg) was separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). After being blocked, the membranes were then incubated with primary antibodies including Bax (1 : 2000, AF0120, Affinity), Bcl-2 (1 : 1000, AF6139, Affinity), cleaved-caspase 3 (1 : 1000, AF7022, Affinity), p62 (1 : 2000, AF5384, Affinity), LC3 (1 : 1000, AF5402, Affinity), Beclin 1 (1 : 1000, AF5128, Affinity), PI3K (1 : 1000, AF6241, Affinity), phospho-PI3K (1 : 1000, AF3241, Affinity), AKT (1 : 1000, AF6264, Affinity), phospho-pan-AKT1/2/3 (Ser473) (1 : 1000, AF0016, Affinity), mTOR (1 : 2000, AF6308, Affinity), phospho-mTOR (Ser2448) (1 : 1000, AF3308, Affinity), and β-actin (1 : 5000, AF7018, Affinity) overnight at 4°C. Subsequently, the membranes were incubated with secondary antibodies (1 : 5000, Kangchen, China) for 1.5 h at room temperature. Protein bands were visualized using an enhanced chemiluminescent (ECL) kit (Beyotime, China). The image intensity was analyzed using the Image J software.
9
Protein Expression Analysis in Kidney Tissue
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Proteins were isolated from kidney tissue and MPC-5, and protein levels were assayed. Briefly, protein extracts were boiled in RIPA buffer (Beyotime, Shanghai, China) and separated by SDS-PAGE electrophoresis, transferred to NC membranes, closed, and then incubated overnight at 4°C with the primary antibodies nephrin (NPHS1 A3048; ABclonal, China), podocin (NPHS2 20384-1-AP; Proteintech, China), and synaptopodin (A8484; ABclonal, China) to detect their protein levels in kidney tissue and MPC-5. Beclin1 (A17028; ABclonal, China), p62 (A11250; ABclonal, China), LC3B (A19665; ABclonal, USA), Phospho-mTOR-S2448 (AP0094, ABclonal, China), mTOR (AF6308, Affinity, China), Phospho-ULK1-S757 (AP0736, ABclonal, China) and ULK1 (AF4687, Affinity, China) antibodies were assessed to detect their protein levels in MPC-5. GAPDH (AC002; Abclonal, China) or Tubulin (A17913; Abclonal, China) were used as reference proteins. The blots were then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and visualized using the Bio-Rad Gel Doc EZ imaging system (Bio-Rad Laboratories, Hercules, CA, USA) and enhanced chemiluminescence reagents (Millipore, Boston, MA, USA). All western blot analyses were performed ≥3 times and protein bands were quantified using the ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA).
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