Fig 1: Identification of kinases responsible for SFL phosphorylation.(A) Schematic diagram of three AirID constructs used for PDB. (B) Working flow of the proximity biotinylation for identifying kinases responsible for SFL phosphorylation. WB, Western blot. (C) The expression plasmids (A) were transfected into HEK293T cells, and 0.5 μM biotins were supplemented in the cell culture to promote biotinylation. The cells were harvested and lysed 16 hours posttransfection and analyzed by Western blot. Input: proteins before SA magnetic bead enrichment. Output: proteins after SA magnetic bead enrichment. Representative of three independent experiments. (D) LC-MS/MS analysis of SA magnetic bead–enriched samples from three independent biological replicates. Gray dots were proteins detected in both the 10- and 35-nm groups; black dot was SFL, and red dots were Ser/Thr kinases with expression levels at least fourfold higher than the AirID group (both 10 and 35 nm), among which six kinases (indicated with blue fonts) were selected for further analysis. FC, fold change. (E) Left: The purple and blue circles represent Ser/Thr kinases with expression levels up-regulated at least fourfold compared to the AirID group in the 10- and 35-nm subgroups. The green circle includes 58 kinases predicted by the PhosphositePlus software. Right: Six Ser/Thr kinases were selected because they represented the consensus of three cycles. (F) Phos-Tag PAGE analysis of in vitro kinase assays. Six selected kinases were tested for the ability to phosphorylate SFL. Phosphorylation species are marked by red numbers. Representative of two independent experiments. (G) Identification of phosphorylation sites in SFL from the marked bands in (F) by LC-MS/MS. (H) Expression of HIV-1 proteins in HEK293T cells with knockdown of EEF2K, NEK9, or PBK genes in the presence or absence of SFL. Representative of two independent experiments.