We washed the cells cultured in 6-well plates (Corning Life Sciences, USA) three times in PBS and lysed them on ice in RIPA buffer, protease-inhibitor cocktail, and phosphatase-inhibitor (APExBIO, USA) solution. They were then mechanically broken down using a syringe. The suspension was centrifuged (4 ℃) and the supernatant was collected. An enhanced BCA protein assay kit (Beyotime Biotechnology, China) was used to determine the protein concentration of the supernatant. Proteins (50 μg) were separated on 10% SDS–polyacrylamide gels and transferred onto PVDF membranes (Millipore, USA). The PVDF membranes were blocked with 5% skim milk in TBS, then incubated with primary antibodies (overnight, 4 °C). After three washings in TBS-T, the membranes were incubated (25 °C, 1 h) with horse radish peroxidase-conjugated secondary antibodies (1:3000; Signalway Antibody, USA) and exposed to immobilon western chemilum hrp substrate (Millipore, USA). To confirm equal protein loading, we used anti-β-actin antibodies (1:2000, Signalway antibody, USA) to re-probe. The antibodies used are listed in Additional file 1 : Table S2.
Horse radish peroxidase conjugated secondary antibodies
Manufactured by Signalway Antibody
Sourced in United States
Horse radish peroxidase-conjugated secondary antibodies are a type of laboratory reagent used in various immunoassay techniques. They serve as a detection tool, binding to and labeling primary antibodies, allowing for their visualization and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Apexbio (1 mentions)
Phosphatase inhibitor, by Apexbio (1 mentions)
Immobilon western chemilum hrp substrate, by Merck Group (1 mentions)
6 well plate, by Corning (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Anti hdac3, by Cell Signaling Technology (1 mentions)
Pan anti acetylated lysine, by PTM Biolabs (1 mentions)
Pan anti crotonylated lysine, by PTM Biolabs (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Ecl detection reagent, by Merck Group (1 mentions)
Anti cd31, by Abcam (1 mentions)
Rabbit anti collagen 1 clone e8f4l, by Cell Signaling Technology (1 mentions)
Rabbit anti fibronectin clone epr23110 46, by Abcam (1 mentions)
Mouse anti stat3 clone 124h6, by Cell Signaling Technology (1 mentions)
Rabbit anti p stat3 tyr705 clone d3a7, by Cell Signaling Technology (1 mentions)
3 protocols using horse radish peroxidase conjugated secondary antibodies
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Protein Expression
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Total proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (PVDF; MilliporeSigma) for Western blotting. The PVDF membranes were blocked with 5% milk for 1 h at room temperature. After treatment, the membranes were incubated overnight with the primary antibodies at 4 ℃. The primary antibodies used were as follows: anti-CD31 (1:500; Abcam, Cambridge, UK), anti-α-smooth muscle actin (SMA; 1:2,000; ImmunoWay, Plano, TX, USA), anti-HDAC3 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), pan-anti-acetylated lysine and pan-anti-crotonylated lysine (1:500; PTM Biolabs, Hangzhou, Zhejiang, China), anti-β-actin (1:1,000; CMCTAG, Dover, DE, USA). The membranes were then washed 3 times for 10 min with Tris-buffered saline Tween-20 (TBST) buffer and incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000; Signalway Antibody, Baltimore, MD, USA) for 2 h at room temperature. The membranes were then washed 3 times for 10 min in TBST before being incubated with enhanced chemiluminescence (ECL) detection reagent (MilliporeSigma) and scanned using an ECL detection system.
3
Western Blot Protein Expression Analysis
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Protein samples were separated by SDS-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA). After being blocked, the membranes were incubated with rabbit anti-collagen I (clone E8F4L, catalog 72026, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-fibronectin (clone EPR23110-46, catalog ab268020, Abcam, Cambridge, UK), rabbit anti-α-SMA (clone EPR5368, catalog ab124964, Abcam, Cambridge, UK), rabbit anti-SOCS3 (polyclonal, catalog AF8025, Beyotime, Shanghai, China), mouse anti-STAT3 (clone 124H6, catalog 9139, Cell Signaling Technology, Danvers, MA, USA), or rabbit anti–p-STAT3 (Tyr705) (clone D3A7, catalog 9145, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Equal protein loading was confirmed by incubation with mouse anti–β-tubulin (clone 5G3, catalog 44032, Signalway antibody, Greenbelt, MD, USA). Horseradish peroxidase-conjugated secondary antibodies (Signalway antibody, Greenbelt, MD, USA) were incubated with the membranes at room temperature for 1 h. Finally, the bands were visualized with electrochemiluminescence (Merck Millipore, Burlington, MA, USA) and analyzed by ImageJ software 1.46r.
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