Fig. 3

FBW7 interacts with NFATc1 and promotes its Lys63-linked polyubiquitylation. a and b HeLa cells were subjected to immunoprecipitation using anti-IgG, anti-FBW7 or anti-NFATc1 as indicated, and were analyzed by Western blot according to standard procedures (n = 3). c FBW7 was depleted in HeLa cells using FBW7 siRNA. The depletion of FBW7 did not affect NFATc1 protein stability (n = 3). d Transfection of FBW7-expressing plasmid in HeLa cells for 24 h and then analyzed by Western blot (n = 3). e Wild-type and FBW7-depleted HeLa cells were transfected with HA-Ub plasmids for 24 h and MG132 was added for another 4 h, then the cell lysates were subjected to IP using an anti-NFATc1 antibody followed by Western blot analysis (n = 3). f Wild-type and FBW7-depleted HeLa cells were transfected with HA-Ub (Lys48, Lys63 only) as indicated and immunoblotted with anti-HA antibody to detect ubiquitylated NFATc1 (n = 3)