Supersignal west pico plus chemiluminescence substrate

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).

HEK293 cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS, 1% penicillin, and 1% L-glutamine (Biological Industries), transfected with AAV. Syne4^WD, AAV. Syne4^AA, or empty FLAG control plasmids, using jetPEI (Polyplus) according to manufacturer’s instructions and harvested 48 h after transfection. One 10 cm plate per condition was lysed 1 ml RIPA buffer (Sigma) and Halt Protease Inhibitor Cocktail (Thermo). Tubes were placed on an end-over-end shaker for 1 h at 4°C followed by centrifugation at 16,000 g for 15 min at 4°C. 20 µl of the resulting supernatant was used as input and the rest was incubated with EZview anti-FLAG M2 beads (F2426, Sigma) for 1-2 h at 4°C on an end-over-end shaker. Beads were then washed and eluted in sample buffer according to manufacturer’s instructions and loaded into a 10% SDS-PAGE gel. Samples were then transferred onto a nitrocellulose membrane which was then blocked in 3% skimmed milk (BD Difco). Membranes were then subjected to immunoblotting. Kif5b was detected by anti Kif5b (Abcam, ab167429). FLAG was detected using rabbit anti DDDDK antibody (Abcam, ab205606). Blots were visualized using anti rabbit HRP antibody (Cell Signaling Technologies, 7074) and SuperSignal West Pico PLUS Chemiluminescence Substrate (Thermo Scientific).