To begin, obtain the HEK293T cells expressing RapR-Shp2 and perform immunoprecipitation with Protein G-Sepharose. After immunoprecipitation, wash the sepharose two times each with 0.5 milliliters of lysis buffer, followed by 0.5 milliliters of wash buffer. Remove as much buffer as possible after the final spin.
Then add 40 microliters of the phospho-paxillin in imidazole buffer mixture to each sample with or without one micromolar rapamycin. Flick the tube gently and immediately place it in the heating shaker for 40 minutes at 32 degrees Celsius. Set the heating shaker to 1000 revolutions per minute to ensure full agitation of the sample.
To stop the reaction, add 40 microliters of 2X Laemmli buffer to each sample and incubate them at 95 to 100 degrees Celsius for five minutes. After the samples cool down, load 15 microliters of each sample onto a four to 15%gradient SDS polyacrylamide gel. Run a standard Western blot protocol using PVDF membranes.
Finally, blot using anti-FLAG antibody to detect the RapR-Shp2 construct and anti-phospho-paxillin to detect phosphorylated paxillin in the samples. Active RapR-Shp2 showed no phospho-paxillin, similar to the constitutively active Shp2, indicating retained full activity. Inactive RapR-Shp2 and dominant negative Shp2 showed similar phospho-paxillin levels, indicating no activity when inactive.
