Plasma membrane fraction from whole-cell lysate was isolated using the Minute Plasma Membrane Protein Isolation Kit according to the manufacturer’s instructions (Invent Biotechnologies, SM-005-P). Briefly, whole-cell lysates were prepared in buffer A (provided by the manufacturer) by vortexing three times for 5 s each and incubating on ice for 20 min followed by centrifugation at 700g for 1 min to separate pellet containing nuclei and larger debris. The supernatant was transferred to a fresh microfuge tube and centrifuged at 4°C for 1 hour at 16,000g. The supernatant containing the cytosolic fraction was transferred to a new tube, and the pellet containing total membrane fraction (organelle membrane and plasma membrane) was isolated. For isolation of plasma membrane fraction from total membrane fraction, the pellet containing total membrane fraction was thoroughly resuspended in 200 ml of buffer B (provided by the manufacturer) by repeated pipetting followed by centrifugation at 8000g for 20 min at 4°C. The pellet containing organelle membrane proteins was isolated. The supernatant was transferred to a fresh microfuge tube to which 1.6 ml of cold 1× PBS was added, mixed, and centrifuged at 16,000g for 1 hour. The pellet containing plasma membrane proteins was resuspended in Minute Denaturing Protein Solubilization reagent (provided by the manufacturer) for SDS-PAGE and immunoblot analysis. Purity was verified by positive expression of E-cadherin but negative expression of early endosome antigen 1.

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