A00702

Membrane and cytosolic proteins were isolated following published protocols with modifications^{83 (link),84 (link)}. Briefly, the same dry weight of titan cells (CUX1331, CRK1^∆PESTP_GDP1-GPA1:FLAG) and typical cells (CUX1197, crk1∆ P_GDP1-GPA1:FLAG) were used for protein extraction. CUX1331 and CUX1197 were harvested and stored at −80 °C after culturing in titan cell inducing condition for 3 days. Samples were lyophilized and the same dry weight of lyophilized samples were subsequently pulverized with a ceramic mortar and a pestle and resuspended in 1.5 ml lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10 µM PMSF and 1× EDTA-free protease inhibitor). Cells were centrifuged at 2000 × g for 10 min at 4 °C to remove non-lysed cells from total cells lysates. The supernatant was transferred to a new tube and separated into a soluble and pellet fraction by centrifugation at 25,000 × g for 60 min at 4 °C. The insoluble pellet was resuspended in 0.1 ml lysis buffer with 1% Triton X-100 to extract membrane proteins. 10 µl of soluble cytosolic proteins and 5 µl of membrane proteins were analyzed by western blotting with FLAG antibody (Genscript, A01868, 1:2000) and actin antibody (Genscript, A00702, 1:5000), marker of cytoplasm.