Co‐immunoprecipitation and ubiquitination assays were carried out as described previously.23 (link) Briefly, the cell lysates were blocked with 40 μl Protein A + G agarose beads (Beyotime Biosciences) at 4°C for 2 hours and immunoprecipitated with 4‐10 μg mouse primary anti‐p53 (#60283‐2‐Ig, Proteintech), mouse primary anti‐FLAG (#HT201‐01, TransGen Biotech) or rabbit primary anti‐p53 (#10442‐1‐AP, Proteintech) antibodies overnight at 4°C. The immune complexes were captured by 30 μl Protein A + G agarose beads and visualized via Western blotting.
For the ubiquitination assay, A549 cells were transfected with specified siRNA or plasmids for 48 hours and then treated with MG132 (MedChemExpress, Monmouth Junction) for 24 hours before they were collected and lysed.
After centrifuging, the supernatant was immunoprecipitated using an anti‐p53 (1:1000, #10442‐1‐AP/#60283‐2‐Ig, Proteintech) antibody overnight at 4°C. Samples were then incubated with Protein A + G agarose (Beyotime Biosciences) and washed there times in NP‐40 lysis buffer. Agarose and 20 μg of the protein immunocomplex were immunoblotted using an anti‐HA antibody (1:1000, #51064‐2‐AP, Proteintech).
For the ubiquitination assay, A549 cells were transfected with specified siRNA or plasmids for 48 hours and then treated with MG132 (MedChemExpress, Monmouth Junction) for 24 hours before they were collected and lysed.
After centrifuging, the supernatant was immunoprecipitated using an anti‐p53 (1:1000, #10442‐1‐AP/#60283‐2‐Ig, Proteintech) antibody overnight at 4°C. Samples were then incubated with Protein A + G agarose (Beyotime Biosciences) and washed there times in NP‐40 lysis buffer. Agarose and 20 μg of the protein immunocomplex were immunoblotted using an anti‐HA antibody (1:1000, #51064‐2‐AP, Proteintech).
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