Clx imaging system

Cells were lysed in immunoprecipitation buffer as above, and lysate protein concentrations were quantified by BCA and normalized before addition of 20–40 μl antibody-conjugated beads (GFP, myc, HA, or V5, listed above, or Dynabead-conjugated anti-TRIM1). Immunoprecipitations were performed at 4°C for 2–12 h. Beads were washed ≥3× in wash buffer (50 mM Tris, pH 7.5, 150–500 mM NaCl, and 1 mM EDTA), and bound proteins were eluted by heating at 70–95°C in 40–80 μl 4× NuPage LDS loading buffer with 5% β-mercaptoethanol. Samples were run on NuPage Novex minigels, either 4–12% Bis-Tris or 3–8% Tris-acetate, and transferred onto PVDF membrane with the Genscript eBlot L1 transfer system and blocked with LI-COR Intercept TBS blocking buffer. All imaging and quantification of immunoblots was performed using a LI-COR Odyssey CLx imaging system and Image Studio Lite software v5.2. All quantification of LRRK2 or phospho-LRRK2 protein levels was performed using 3–8% Tris-acetate gels.

Cell lysates were created via radio immunoprecipitation assay (RIPA) buffer and were separated by Tris-glycine PAGE, semi-dry transferred to nitrocellulose membranes and visualized using the Licor CLX imaging system. Unaltered complete blots are provided as a supplement with cropping regions indicated. Bands outside of the cropping region are considered non-specific due to their random appearances when conditions were repeated independently.

pVAX1-D3ME or pVAX1 was transfected into Vero cells using Lipofectamine 2000 (Thermo Fisher Scientific, USA). Seventy-two hours after transfection, the supernatant and lysate were collected and fractionated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane according to the manufacturer's recommendation (Millipore, USA). The membrane was blocked with 5% bovine serum albumin for 2 h and incubated with anti-flavivirus E protein monoclonal antibody (1:50, D1-4G2-4-15 hybridoma supernatant, ATCC HB-112) at 4°C overnight, and then probed with goat anti-mouse HRP secondary antibody (1:4,000, abcam, China) for 1 h. Blots were developed with chemiluminescent HRP substrate peroxide solution mix (Millipore, USA) and visualized with Li-Cor Odyssey CLx imaging system.

Cells were lysed with RIPA on ice for 20 min. After centrifuging, the total protein was quantified by Bicinchoninic acid (BCA). And then, the protein was separated on SDS–PAGE gels. After transferring onto NC membranes., the membranes were blocked with defatted milk for 3 h and then incubated with different primary antibodies at 4 °C overnight. After washing with TBST 3 times, the membranes were incubated with the Secondary Antibodies for 1 h. proteins were visualized via Licor Odyssey® CLx Imaging System.