Immunoprecipitation of endogenous MEK1 or FLAG-tagged BRAF/KSR1 to evaluate the effect of different MEKi on MAPK complex assembly

HEK293T cells (350,000) were seeded in a six-well, flat-bottom tissue culture-treated plate. After 24 h, cells were transfected with either FLAG-tagged KSR1 or FLAG-tagged BRAF constructs at a concentration of 1 μg ml^–1 in combination with 9 μg ml^–1 carrier DNA for the FLAG pulldowns or 0.5 μg ml^–1 in combination with 9 μg ml^–1 carrier DNA for the endogenous MEK1 pulldown. Fugene HD was added to both conditions. Twenty-four hours after transfection, cells were treated with trametinib, Tram-bo, trametiglue or CH5126766, each at 1 μM, at a final DMSO concentration of 0.1% in the medium. After 24 h, cells were washed with sterile 1× PBS and collected in Pierce IP lysis buffer (Thermo Fisher, 87787) supplemented with HALT phosphatase and protease inhibitor cocktail and quantified and normalized using an RC DC Protein Assay kit (Bio-Rad, 5000122). Dynabeads M-280 sheep anti-rabbit IgG (Invitrogen, 11203D) were preconjugated to 5 μg per sample of anti-MEK1 (EMD Millipore, 07–641), anti-FLAG (Cell Signaling Technologies, 14793) or normal rabbit IgG antibody (Cell Signaling Technologies, 2729) by rotating for 1 h at room temperature. Next, the conjugated mixtures were washed thoroughly with PBS (0.1% Tween 20) three times using a magnetic rack. For endogenous MEK1 IP, 100 μg of total cell lysate was mixed with preimmobilized anti-MEK1 conjugated to anti-rabbit magnetic Dynabeads. For FLAG IP, 100 μg of total cell lysate was mixed with preimmobilized anti-FLAG conjugated to anti-rabbit magnetic Dynabeads. As a control for both conditions, 100 μg of total cell lysate was mixed with preimmobilized normal rabbit IgG conjugated to anti-rabbit Dynabeads (5 μg per 100 μg of cell lysate). Samples were incubated for 1 h at 4 °C on a rotator and were subsequently washed three times with PBS (0.1% Tween 20). Protein lysates were eluted off beads by adding 70 μl of 2× Laemmli buffer to each sample and heating to 80 °C for 5 min. Eluted proteins were analyzed via western blotting. The following primary antibodies were used for western blotting: anti-BRAF (Santa Cruz Biotechnology, sc-5284; 1:1,000), anti-MEK1 (Cell Signaling Technologies, 2352; 1:1,000) and anti-FLAG (Cell Signaling Technologies, 8146; 1:1,000). The secondary antibody used was anti-mouse horseradish peroxidase (Cell Signaling Technology, 7076S; 1:10,000)