After treatment with
drugs, H9C2 cells and RBMEC were collected separately and then washed
twice with PBS before being lysed using a RIPA buffer. Total protein
was separated by SDS-PAGE and transferred onto a PVDF membrane. After
the transfer of proteins, a blocking step was carried out using a
1× TBST solution fortified with nonfat milk powder (5%) at room
temperature for 2 h. Subsequently, the samples were incubated with
the primary antibody overnight at 4 °C. On the subsequent day,
the membrane was washed three times with TBST, followed by incubation
with the corresponding secondary antibody (mouse or rabbit) at room
temperature for 1 h. Finally, the images were captured using a chemiluminescence
imaging system (Azure Biosystems, Dublin, USA) and subsequently analyzed
with ImageJ software.
drugs, H9C2 cells and RBMEC were collected separately and then washed
twice with PBS before being lysed using a RIPA buffer. Total protein
was separated by SDS-PAGE and transferred onto a PVDF membrane. After
the transfer of proteins, a blocking step was carried out using a
1× TBST solution fortified with nonfat milk powder (5%) at room
temperature for 2 h. Subsequently, the samples were incubated with
the primary antibody overnight at 4 °C. On the subsequent day,
the membrane was washed three times with TBST, followed by incubation
with the corresponding secondary antibody (mouse or rabbit) at room
temperature for 1 h. Finally, the images were captured using a chemiluminescence
imaging system (Azure Biosystems, Dublin, USA) and subsequently analyzed
with ImageJ software.