Western Blot
The cells were washed 3 times with a 1 × PBS buffer, then were lysed in
an ice-colded RIPA extract buffer (FUDE, Cat: FD008, Jiangsu, China)
supplemented with a mixture of protease inhibitors (SIGMA, Cat:
#021M8200, Guangzhou, China). The extracted proteins were separated by
SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane
(Bio-Rad, Cat: #1620177, USA). After blocking with a TBS/T buffer
(0.1%, containing 5% bovine serum albumin) for 30 min at room
temperature, the membrane was incubated with different primary
antibodies at 4 °C for overnight, including SGLT2 antibody (Abcam,
Cat#ab37296), and Actin (Cell Signaling Technology Cat# 4970). The
membrane was washed with the TBS/T buffer four times to remove the
unbound antibody and then incubated with the HRP-conjugated secondary
antibody (Cell Signaling Technology). Protein bands were visualized with
an ECL kit (Millipore, Cat: WBKLSO500). The densitometry analysis was
performed using the Quantity One Software (Bio-Rad, CA, USA) relative to
the loading control.