Pierce classic magnetic ip and co ip kit

For the Co-IP assay, the Pierce Classic Magnetic IP and Co-IP Kit from Thermo Scientific (Dagma, Carcavelos, Portugal) were used. A total of 5.0 × 10⁵/flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 µg of total protein was incubated with 10 µL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C. The immunoprecipitation of the immunocomplexes was performed using magnetic beads. The Western blot analysis was performed, as in Section 4.6, for detection of p53 and MDM2 in whole-cell lysate (input) and in immunoprecipitated proteins. GAPDH was used as loading control.

Co‐IP was performed using the Pierce Classic Magnetic IP and Co‐IP Kit (Thermo Scientific, Dagma, Carcavelos, Portugal), as described (Soares et al., 2015a). Detection of p53, MDM2, MDMX, and GAPDH was performed by western blot.