Gtx115044

HEK 293 cells were grown in 96- well or 384-well plates and were transfected with PEI as aforementioned. The cells in each well were lysed in 120 µl (96 well) or 80 µl (384 well) TNE buffer (Tris pH 7.5 20 mM, NaCl 150 mM, EDTA 2 mM and Triton X-100 0.5% supplemented with protease inhibitors). Aliquots of lysates (20 µl) were incubated for 3 h at 4 °C in a well of a 384 well Lumitrac plate (Greiner Bio-One) that was coated with α-FLAG antibody (20 µl/well with 1:100 dilution) and blocked with BSA. After three times thorough wash with phosphate buffer saline supplemented with 0.05% Tween 20 (PBST), the plate was incubated with HRP-conjugated α-HA antibody (GeneTex GTX115044, 1:5000 dilution) for 1 h at room temperature. The plate was washed three times with PBST followed by chemiluminescence reading using SuperSignal ELISA Pico substrate (ThermoFisher).

The mouse BicD2 cDNA construct was purchased from Sino Biological (MG5A2126-NY, GenBank Accession No.: NM_001039179.2) and was cloned to pCAGGS construct, which was a gift from Phil Sharp (Addgene plasmid # 41583) [17 (link)] with an HA tag. US2-GFP construct was a generous gift from Dr. Jenn-Yah Yu (National Yang-Ming University Taiwan). The sources of the primary antibodies used in this study are listed as follows: Rabbit anti-Ki67 (AB9260, Millipore), Rabbit anti-Pax-6 (#901301, BioLegend), anti-Cux1 antibody (sc-13,024, Santa Cruz), Rabbit anti-NeuN (ABN78, Millipore), Rabbit anti-BICD2 (ab117818, Abcam), Rabbit anti-HA (GTX115044, Genetex), Mouse anti-HA (66006–1-Ig, Proteintech), Mouse anti β-actin (66009–1-Ig, Proteintech), Mouse anit-α-tubulin (66031–1-Ig, Proteintech), Mouse anti-lamin A/C 636 (sc-7292, Santa Cruz), Mouse anti-Dynein heavy chain C-5 (sc-514,579, Santa Cruz), Mouse anti-Dynein light intermediated chain clone 74.1 (MAB1618, Millipore).

The following primary antibodies were used: rabbit anti-JEV-NS4A (GeneTex, GTX132028), rabbit anti-PINK1 (Abcam, ab23707), mouse anti-PINK1 (Abcam, ab75487, ab186303), rabbit anti-TOMM20 (Santa Cruz, sc-11415), mouse anti-COX2 (Santa Cruz, sc-514489), rabbit anti-COX4 (CST, no. 4844), rabbit anti-mitofusin-1 (CST, no. 14739), rabbit anti-mitofusin-2 (CST, no. 11925), rabbit anti-DRP1 (CST, no. 8570), rabbit anti-p-DRP1 (Ser 616; CST, no. 4494), rabbit anti-p-PARKIN (S65; CST, no. 36866S), mouse anti-PARKIN (CST, no. 4211S), rabbit anti-FACL4 (ab155282), rabbit anti-OPA1 (CST, no. 67589), goat anti-SERCA2 (Abcam, ab183531), rabbit anti-β-actin (CST, no. 4970), rabbit anti-HA (GeneTex, GTX115044), rabbit anti-GAPDH (GeneTex, GTX100118), rabbit anti-GFP (Cloud-clone Corp., PAD025Ge07), and GFP-trap antibody (Chromotek, GTA-20 GFP-TRAP). The horseradish peroxidase (HRP)-conjugated secondary antibodies used were donkey anti-mouse and anti-rabbit antibodies (Jackson Immunochemicals, 711-035-152 and 715-035-150). The secondary antibodies used in immunofluorescence experiments were chicken anti-mouse antibody-Alexa fluor 488 and goat anti-rabbit antibody-Alexa fluor 568 (NextGen Life Sciences, A21200 and A11011).