Tissues or cells were lysed in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors. Protein lysates were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidine difluoride membranes. Proteins were detected by standard Western blotting procedures. Antibodies were diluted 1:1000 for BMAL1 (Abcam, ab93806), CRY1 and CRY2 (98 (link)), c-MYC (Abcam, ab32072), HSF1 (CST-12972), KRASG12D (CST-14429), p-ERK1/2 (CST-4370), and ERK1/2 (CST-4695); 1:2000 for REV-ERBα (33 ), DNAJB1 (Enzo, ADI-SPA-400), and HSP90AA1 (GTX109753); 1:10,000 for LAMIN A (Sigma-Aldrich, L1293); 1:50,000 for ACTIN (Sigma-Aldrich, A1978). Imaging and band quantification were carried out using ChemiDoc XRS+ System (Bio-Rad).
Protocol detail
Western Blot Analysis of Cellular Proteins
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Actin
Antibodies
Buffer
C myc
Cells
Dnajb1
Erk1
Hsp90aa1
Inhibitors
Lamin a
Membranes
Phosphatase
Protease
Proteins
Radioimmunoprecipitation assay
Sds polyacrylamide gel electrophoresis
Tissues
Corresponding Organization : Scripps Research Institute
Other organizations : University of Rochester Medical Center, Salk Institute for Biological Studies
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Variable analysis
independent variables
- Protein lysates
- BMAL1 protein levels
- CRY1 and CRY2 protein levels
- C-MYC protein levels
- HSF1 protein levels
- KRASG12D protein levels
- P-ERK1/2 protein levels
- ERK1/2 protein levels
- REV-ERBα protein levels
- DNAJB1 protein levels
- HSP90AA1 protein levels
- LAMIN A protein levels
- ACTIN protein levels
- Tissues or cells were lysed in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors.
- Protein lysates were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidine difluoride membranes.
- Proteins were detected by standard Western blotting procedures.
- None specified
- None specified
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