Figure 5: Murine free Isg15 does not interact stably with Usp18.
From: ISG15 deficiency and increased viral resistance in humans but not mice
(a) HEK293T cells were transfected with a human USP18 expression vector (0.5 μg) alone or with increasing amounts of the human Flag-ISG15ΔGG construct. 48 h post transfection, cell lysates were analysed by western blot with antibodies against USP18 and Flag. (b) Cells were transfected with human USP18 (0.5 μg) alone or with increasing amounts of human ISG15 or GFP expression vectors. 48 h later lysates were analysed with the indicated antibodies. (c) Cells were transfected with human Flag-ISG15ΔGG (3 μg) alone or with human USP18, as indicated (1.5 μg). NEM (10 mM) was added to the lysis buffer (lane 3). Lysates were subjected to IP with USP18 antibodies. Lysates and co-IP eluates were analysed with antibodies against USP18 and Flag. (d) Left panel: cells were transfected with human Flag-ISG15 (1.5 μg), and either human USP18 WT or USP18 C64S (1.5 μg). 48 h later, lysates were subjected to anti-USP18 IP. Lysates and co-IP eluates were analysed with antibodies against USP18 and Flag. Right panel: cells were cotransfected with Flag-ISG15 (1.5 μg) and USP18 C64S (1.5 μg). NEM (10 mM) was added to the lysis buffer (lane 3). Lysates were subjected to anti-USP18 IP. Lysates and co-IP eluates were analysed with antibodies to USP18 and Flag. (e) Cells were cotransfected with human or murine USP18-V5 and human or murine Flag-ISG15. 48 h later, lysates were subjected to co-IP with anti-V5 antibodies. Co-IP eluates (top panels) and total lysates (bottom panels) were analysed with the indicated antibodies. (f) Cells were transfected with untagged murine Usp18 (0.5 μg) alone or with increasing amounts of murine Flag-Isg15. Lysates were analysed with antibodies to murine Usp18, Flag and Akt. (g) As in f, except that HA-tagged murine Usp18 was transfected and detected with HA antibodies. (h) Cells were transfected with human USP18-V5 or murine Usp18-V5 Usp18 (500 ng) alone or with the indicated amount of human Flag-ISG15 or murine Flag-Isg15. 48 h later, cell lysates were analysed by western blot.
