FLAG pulldown assays

To assess protein complex formation of heterologously expressed proteins, coimmunoprecipitations were performed in 293T cells. Cells were seeded at a density of 2 × 10⁵ cells/mL in a 6-well plate. For assessing binary CALR^del52-MPL complex formation, DNA-cationic lipid mixtures were created by combining 1.25 μg of pCMV-SPORT6-CALR variant-expressing plasmid, 1.25 μg of pLeGO-iT2-hMPL, and 7.5 μL TransIT-LT1 transfection reagent (Mirus) in 250 μL of media-free DMEM. For assessing ternary CALR^del52-MPL complexes with 2 different tagged versions of CALR^del52, 0.625 μg of each pCMV-SPORT6-CALR variant-expressing plasmid was used and mixed with 1.25 μg of pLeGO-iT2-hMPL and 7.5 μL TransIT-LT1 transfection reagent in 250 μL media-free DMEM, as discussed previously. Mixtures were allowed to sit at room temperature for 15 minutes, and then added dropwise to 293T cells. After 24 hours, transfected 293T cells were harvested, washed with PBS twice, and then cells were collected and resuspended in 450 μL NP40 lysis buffer and incubated on ice for 30 minutes. Lysates were cleared by centrifugation at 17000g for 10 minutes at 4°C, and 10% (usually ∼50 μL) was set aside as the total lysate “input” fraction and the remainder used for pulldown assays. Protein G bead-antibody mixtures were generated by initially washing 50 μL of magnetic Protein G Surebeads (BioRad Laboratories) per sample with PBS with 0.1% Tween-20 [PBST]) 3 times using a magnet. One microliter of FLAG M2 antibody (Sigma) was diluted with PBST and added to the beads and incubated for 15 minutes with inversion at room temperature. FLAG M2-Protein G beads were washed with PBST and incubated with lysate supernatants for 1 hour at room temperature and mixed by inversion. Lysate-bead mixtures were washed 3 times by magnetic sorting, and beads were resuspended in 100 μL of 1X SDS Laemmli buffer and eluted by incubating the beads for 10 minutes at 70°C. Both pulldown fractions and lysates were analyzed by Western immunoblotting, as described in supplemental Information.