Fig. 1: RBM47 is ISGylated at Lys329.

Endogenous RBM47 showed a slower-migrating band (indicated by an arrowhead), which was reduced upon the knockdown of RBM47. RBM47 siRNA#1 (A) and RBM47 siRNA#2 (B), and scrambled siRNA were transfected into A549 cells for 72 h, followed by WB analysis (WB) using antibodies recognized RBM47 and Tubulin. Depletion of ISG15 (C) and HERC5 (D) inhibited ISGylation of RBM47. The slower migrating band of RBM47 (arrowhead) was reduced upon the knockdown of ISG15 or HERC5. ISG15 siRNA (C), HERC5 siRNA (D), and scrambled siRNA were transfected into A549 cells for 72 h, followed by WB analysis using antibodies recognized ISG15, HERC5, Actin, and RBM47. E Endogenous RBM47 is ISGylated. 293 T cells were co-transfected with plasmids expressing components of the ISGylation conjugation system (ISG15 conj.), including UBE1L, UBCH8, HERC5, and FLAG-ISG15. 48 h after transfection, cell lysates were subjected to immunoprecipitation (IP) using anti-FLAG magnetic beads followed by WB analysis using antibody-recognized RBM47 (upper panel). And the cell lysates were analyzed by WB using antibodies recognized RBM47 and ISG15 (lower panel). F The ISGylation of RBM47 was abolished by mutating all lysines to arginine (RBM47-KR mutant). 293 T cells were transfected with plasmids expressing ISGylation modification enzymes as in (E), plus plasmids expressing wild-type RBM47 (line 1) and RBM47-KR mutant (line 4) as indicated. In addition, G538A mutant (line 2) and RBM47-CA (all cysteine to arginine) mutant (lane 3) were included as controls. 48 h after transfection, cell lysates were subjected to IP using anti-FLAG magnetic beads, followed by WB analysis using antibody recognized RBM47. And the cell lysates were analyzed by WB using antibodies as indicated. G The ISGylation of RBM47 was reduced by mutation of lysine 329 to arginine (K329R). The experiments were down as in (F), except that RBM47 and a series of RBM47 lysine (K) to arginine (R) substitution mutants (including K317R, K321R, K329R, and K374R) were expressed. H K329 is the acceptor site for ISGylation. In the RBM47-KR mutant, each arginine mutation was reversed back to lysine one at a time. The experiments were down as in (F), except that RBM47 and KR mutant with R to K back mutation (including R317K, R321K, R329K, and R374K) were expressed.