Western Blot for Transfection Efficiency

We performed western blotting to detect the transfection efficiency of the different variants of each transporter with same method as in our previous paper [28 (link)]. We prepared a transiently transfected cell monolayer, as shown above. We collected the cells with a scraper into phosphate-buffered saline (PBS, pH = 7.4) and centrifuged them at 150x g for 5 min. After this step, we lysed the cells with a 10% SDS and 9% protease inhibitory cocktail (Merck, cat I3786). We resuspended cells in lysis solution immediately, and then we sonicated the samples with a UP50H (Hielscher) ultrasound homogenizer on ice (25 impulses per sample at 60% amplitude). Afterward, we mixed the samples with 2x Laemmli loading buffer (Merck, cat. S34701) and denatured them at 56°C for 60 min. The samples were loaded on an SDS-PAGE acrylamide gel (5% focusing and 10% separating gel). After separation and blotting on a PVDF membrane, we blocked the membrane with 5% nonfat milk for 1.5 hr and then incubated it with the primary antibody overnight at 4°C. We used the anti-tGFP antibody 1 : 1,000 and anti-CapZ antibody 1 : 1,000. Then, we incubated it with the secondary, HRP conjugated antibody 1 : 7,000 and detected the signal from the SuperSignal West Pico Plus Chemiluminescence substrate (Thermo Scientific, USA). The chemiluminogram was taken using a BioRad Chemidoc touch imaging system (Biorad, USA).