Each human RCC cell line was plated at a density of 25 000 cells/well on a 24‐well cell culture plate and cultured for 24 h. After removing the medium, cellular proteins were extracted with RIPA buffer (Thermo Scientific, Waltham, MA, USA) and separated by SDS‐polyacrylamide gel electrophoresis. Anti‐β‐catenin antibody (Abcam, Cambridge, UK) and anti‐phosphorylated ERK antibody (Cell Signaling Technology, Beverly, MA, USA) were used as primary antibodies. Anti‐SULF‐2 (H2.1) antibody was produced by MBL as described in the above section. Antibody‐bound protein bands were visualized using ECL Advance Western detection reagents (GE Healthcare, Buckinghamshire, UK) and imaged using a ChemiDoc XRS plus system (BIO‐RAD, Hercules, CA, USA). Individual bands were quantified with Image Lab 2.0 software (BIO‐RAD), and normalized against the β‐actin band. Experiments were conducted at least three times.
Ecl advance western detection reagents
Manufactured by GE Healthcare
Sourced in United Kingdom
ECL Advance Western detection reagents are a set of reagents used for the detection and visualization of proteins in Western blot analysis. They provide a chemiluminescent signal that can be detected using a compatible imaging system. The reagents are designed to enable sensitive and accurate protein detection.
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Lab products found in correlation
Chemidoc xrs plus system, by Bio-Rad (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Anti β catenin antibody, by Abcam (1 mentions)
Image lab 2, by Bio-Rad (1 mentions)
Anti phosphorylated erk antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab17942, by Abcam (1 mentions)
Ab62739, by Abcam (1 mentions)
α tubulin sc 5286, by Santa Cruz Biotechnology (1 mentions)
Ab38898, by Abcam (1 mentions)
2 protocols using ecl advance western detection reagents
1
Western Blot Analysis of RCC Cell Lines
2
Quantifying Protein Expression Changes
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Two days after transfection, total proteins were extracted and quantified with a BCA protein assay kit (Pierce, Bonn, Germany). Proteins were separated by 10% SDS/polyacrylamide gels and subsequently transferred to poly(vinylidene difluoride) membranes (Millipore, Bedford, MA, USA). After blocking with 5% non‐fat milk, the membranes were incubated with primary antibodies including SIRT6 (ab62739, Abcam, Cambridge, MA, USA), MMP9 (ab38898; Abcam), extracellular signal‐regulated kinases 1 and 2 (ERK1/2; ab17942; Abcam) and p‐ERK1/2 (no. 4370, Thr202/Tyr204; Cell Signaling Technology, Beverly, MA, USA). The secondary antibodies were obtained from Cell Signaling Technology (nos 7074 and 7076). α‐Tubulin (sc‐5286, Santa Cruz Biotechnology, Dallas, TX, USA) was used as a loading control. The protein bands on the membranes were detected with ECL Advance Western detection reagents (GE Healthcare, Little Chalfont, UK) and visualized with ChemiDoc XRS plus system (Bio‐Rad, Hercules, CA, USA).
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