TRPC5-tranfected 293T cells were incubated with Sulfo-NHS-Biotin [0.5 mg/ml in phosphate-buffered saline (PBS), Aladdin] for 30 min and washed thrice with 50 mM glycine in PBS. Cells were lysed and aliquoted for extraction of plasma membrane proteins and total proteins separately. Biotinylated plasma membrane proteins were captured with streptavidin magnetic beads (Invitrogen). Western blotting was then performed to detect TRPC5 in the plasma membrane and total proteins. The ratio between biotinylated and total TRPC5 proteins was used to represent the level of TRPC5 proteins expressed at the cell surface. TRPC4 constructs were analyzed in the same way. Primary antibodies against TRPC5 and TRPC4 were purchased from Sangon Biotech (Shanghai, China) and Proteintech (Wuhan, China), respectively.

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