Membrane lipid strip

FLAG Purification Kit (CELLMM2, Sigma) was used to purify wild-type and mutant FBXW8 from established HEK293T stable cell lines according to the manufacturer’s instructions. The eluted proteins were filtered and concentrated using a 10-kDa cut-off concentrator (Pierce, 88513).
Membrane Lipid Strip (P-6002, Echelon Biosciences) and PIP Strip (P-6001, Echelon Biosciences) were used to perform binding assays between recombinant proteins and lipids as described previously^{23 (link)}. PtdIns4P-conjugated beads (P-B004, Echelon Biosciences), phosphatidylcholine-conjugated beads (P-B0PC, Echelon Biosciences) and control beads (P-B000, Echelon Biosciences) were used to pull down purified recombinant proteins according to the manufacturer’s instructions. Briefly, 100 μl slurry was pelleted by centrifugation at low speed and then resuspended in 300 μl wash-binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl and 0.25% Igepal). Ten micrograms recombinant wild-type and mutant FBXW8 were added for 3 h incubation. The beads were pelleted and washed with wash-binding buffer six times. At last, the proteins were eluted by boiling the beads at 95 °C for 5 min with 2× Lämmeli buffer and analysed by immunoblotting.

The lipid-binding specificity of NS1 was identified by membrane lipid strip (Echelon Biosciences Inc., p-6002). In brief, the strip was blocked by 3% BSA in TBST. Then the membrane strip was incubated with 0.5 ug Myc-tagged NS1 proteins for 1 h. After three washes, the anti-Myc antibody (1:5,000) was incubated with the strip for 1 h, followed by the secondary antibody incubation for another 1 h. Lipid-bound NS1 proteins were detected by ECL plus western blot system (Perkin Elmer, Waltham, MA, United States).

Lipid arrays (Echelon Biosciences, Salt Lake City, UT, USA) used in this work are PIP Strip (P6001), Membrane Lipid Strip (P6002), Membrane Lipid Array (P6003), PIP Array (P6100), and Sphingo Array (S6001). After saturation (3% bovine serum albumin and 0.1% Tween 20 in TBS buffer), membranes were incubated overnight with 5 mL of the protein of interest dissolved in the same buffer. After 4 washes with 0.1% Tween 20 in TBS buffer, the lipid-bound protein was detected upon 1-h incubation with the relevant HRP-labelled anti-tag antibody, followed by a further 4 washes. Detection antibodies were visualised and quantified using Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer, Waltham, MA, USA) and a ChemiDoc imager (Bio-Rad, Hercules, CA, USA). The software automatically checks saturation and auto-scaled images to optimise signal/noise.