Odyssey ir imager

For the detection of palmitoylated Yck2, 30 OD units of yeast cells at OD = 1 were lysed by glass bead disruption in 600 µl ABE lysis buffer (LB) (50 mM Tris buffer pH7.4, 5 mM EDTA, 150 mM NaCl and 10 mm N-ethylmaleimide (NEM)). The lysate was centrifuged for 4 min at 300 g, and Triton X-100 was added to 1.7% final concentration to the supernatant. The samples were incubated with rotation for 1 h at 4°C. The proteins were precipitated with chloroform:methanol. The pellets were air-dried and resuspended in 30 µl of buffer SB (4% SDS, 50 mm Tris–HCl pH 7.4, 5 mm EDTA, 10 mm NEM). In total, 120 µl of LB with 1 mm NEM were added, and the samples were incubated overnight at 4°C. The rest of the protocol was performed exactly as described in [61 (link)] but using the whole amount of protein for each sample.
Tf-Yck2 was detected using an anti-FLAG monoclonal antibody (Sigma, St Louis, Missouri, USA, product code F3165). Anti Vac8 polyclonal antibody was a gift from Dr Christian Ungermann. The blots were probed using secondary antibodies IRdye680 Goat anti-rabbit LI-COR. Product code: 926-68071 or IRdye800 Goat anti-mouse immunoglobulin G (IgG). LiCor. Product code 926-32210 (Licor Biosciences) at a 1/20 000 dilution and then scanned using an Odyssey IR imager (Licor Biosciences).

Virus-infected brainstem slices (200 μm), displaying CNPase-GFP+cells, were homogenized using a protein extraction buffer (ThermoFisher Scientific) as well as protease inhibitor cocktail. The lysates were incubated for 30 min on ice and then centrifuged at 15,000 r.p.m. for 30 min at 4 °C. Supernatants were collected and protein concentrations were estimated using a BCA protein assay kit (Thermo Scientific). Equal amounts of protein were resolved by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and transferred onto polyvinylidine fluoride membrane. The membranes were blocked for 1 h at room temperature and incubated overnight in primary antibody (MBP, 1:1000, BioLegend, SMI-99P and β-actin, 1:1000, Cell signaling, 8H10D10) at 4 °C. Membranes were incubated with IR-conjugated secondary antibodies for 2 h and scanned using Li-COR Odyssey IR imager. MBP band intensities were quantified and normalized to β-actin. Images of Western blots have been cropped for presentation. Full-size western blots with protein ladders (LI-COR, P/N 928-60000) are shown in Supplementary Fig. 7.

For experiments where virion yield was determined, a portion of the cells collected at each time point was resuspended in SDS-PAGE loading buffer. Simultaneously collected culture supernatants were clarified by centrifugation at 1000 rpm for 5 min filtered (0.22 μm), layered over 20% sucrose in 1X PBS and centrifuged at 14,000 rpm in an Eppendorf 5417R microfuge for 2 hours at 4°C. Virion pellets were resuspended SDS-PAGE loading buffer. Cell and virion lysates were separated on 4–12% acrylamide gels (Novex) and proteins transferred to nitrocellulose membranes which were then probed with antibodies against HIV-1 Gag (183-H12-5C, NIH AIDS Research and Reference Reagent Program). The blots were then probed with anti-mouse IgG conjugated to IR Dye800 CW (LiCOR), scanned with a LiCOR Odyssey IR imager and quantified using Odyssey quantification software.

The assays were carried out as described previously (16 (link), 21 (link)). Transfected HEK293 cells were plated into a 96-well poly-d-lysine–coated, black-walled, clear-bottomed plate (Greiner) and assayed 48 h post-transfection. The cells were stained on ice in growth medium (GM: DMEM + 10% fetal bovine serum) for the FLAG epitope applying mouse anti-FLAG-M2 (Sigma, 1:100 in GM), then washed in GM, and stained for 1 h with anti-mouse IRDye800CW goat IgG (Licor, 1:100 in GM). The cells were then fixed, permeabilized and blocked in Odyssey blocking buffer (OBB) before staining at room temperature for 1 h with anti-HA antibody (Immune Systems, 1:1000 in OBB) followed by anti-rabbit secondary IRDye690RD (Licor, 1:500 in OBB). Separate wells were stained with TO-PRO^TM-3-iodide (1:500) to account for cell number. Cell staining was imaged on an Odyssey IR imager and were analyzed using Image Studio Lite version 5.2 (freeware from Licor). Staining intensity was normalized to cell number and background-subtracted before calculating the ratio of surface FLAG:HA total protein staining.