Total protein was collected from SUNE1 and CNE2 using radioimmunoprecipitation (RIPA) containing PMSF (Beyotime, China). Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) was used to separate proteins. During electrophoresis, the proteins were transferred from the gel to a polyvinylidene difluoride (PVDF) membrane. The PVDF membranes were blocked for 15 min in a rapid blocking solution (Beyotime, China) and immunoblotted with the primary antibodies overnight at 4 °C, including anti-YBX1 (Abcam,USA), anti-E-cadherin (CST,USA), anti-Vimentin (CST,USA), anti-N-cadherin (CST,USA), anti-Snail (CST,USA), and anti-β-actin (CST,USA). The next day, the PVDF membrane and the secondary antibody were incubated for 2 h at room temperature. Immune signals were visualized using enhanced chemiluminescent detection reagent (Beyotime, Shanghai, China). Finally, immunoreactive bands were detected by ChemiDoc XRS+ (Bio-Rad, USA).
Enhanced chemiluminescent detection reagent
Manufactured by Beyotime
Sourced in China
Enhanced chemiluminescent detection reagent is a laboratory product designed to facilitate the detection of low-abundance proteins in Western blot analysis. It generates a luminescent signal in the presence of the target protein, allowing for sensitive and quantitative measurement.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xr, by Bio-Rad (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Rapid blocking solution, by Beyotime (1 mentions)
Anti ybx1, by Abcam (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rna extraction kit, by Takara Bio (1 mentions)
Ici182780, by R&D Systems (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Alkaline phosphatase alp activity kit, by Beyotime (1 mentions)
Microbca assay kit, by Beyotime (1 mentions)
2 protocols using enhanced chemiluminescent detection reagent
1
Evaluating Protein Expression in Cancer Cell Lines
2
Icaritin Enhances Osteogenesis in Cells
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Icaritin (>98% purity) was purchased from Shanghai U-sea Biotech Co. Ltd. modified Eagle's medium of alpha (α-MEM) and fetal bovine serum (FBS) were both obtained from Hyclone (Hyclone; GE Healthcare Life Sciences); The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. Dimethyl sulfoxide (DMSO), trypsin and TRIzol® reagent were purchased from Gibco; Thermo Fisher Scientific, Inc.; the alkaline phosphatase (ALP) activity kit, enhanced chemiluminescent detection reagent and micro-BCA assay kit were obtained from Beyotime Institute of Biotechnology. DKK-1 and ICI182780 were purchased from R&D Systems, Inc. The RNA extraction kit was purchased from Takara Bio, Inc. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) primers were obtained from Invitrogen; Thermo Fisher Scientific, Inc. The antibodies used were obtained from Cell Signaling Technology, Inc., unless otherwise indicated. Other reagents used in the experiment were purchased from Sigma-Aldrich; Merck KGaA. Icaritin was dissolved in DMSO and the final concentration of DMSO was 0.05% (v/v). ICI 182780 was dissolved in DMSO and stored at 4°C. Cells were pretreated with ICI 182780 (1 µM) for 30 min at 37°C, followed by icaritin treatment.
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