2.9. Acyl-PEGyl exchange gel shift (APEGS)

The constructed different sIFITMs expressing plasmids were transfected into HEK293T cells in 6 well plates, followed by TEA buffer with 4% SDS for lysate. The protein concentration of cell lysate was determined by BCA. The acyl-PEGyl exchange gel shift (APEGS) assay was carried out in the way as Yokoiet al. described (^{Yokoi et al., 2016}) and modified appropriately according to Kanadome's method (^{Kanadome et al., 2019}). In brief, tris-(2-carboxyethyl) phosphine (TCEP, Thermo) and N-ethyl maleimide (NEM, Wako) was added into the cell lysis successively and incubated with at 25 °C and then recovered twice using Chloroform/Methanol Precipitation (CMppt). Then the recovered protein was dissolved in TEA buffer containing 4 mM EDTA, 4% SDS, and divided into 2 portions, one portion was added 0.75 M neutralized NH₂OH dissolved in TEA buffer and 0.2% Triton X-100, another one portion, as controls, containing TEA buffer and 0.2% Triton X-100. The mixtures above were incubated at 25 °C for 2 h. Then the 5 KDa maleimide-conjugated PEGs (mPEG-5k; NOF) were used for the replacement of S-palmitate of cysteines and incubated at 25 °C for 3 h. Finally, all treated samples including controls and untreated input and internal reference were measured by Western blotting and visualized by fluorescence gel scanning. Image-Pro Plus image software was used to calculate the band intensity for quantitative analysis of S-palmitoylated protein.