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. 2009 Dec 26;285(8):5224–5231. doi: 10.1074/jbc.M109.042812

FIGURE 3.

FIGURE 3.

Cbl proteins mediate NFATc1 ubiquitination in a Src kinase-dependent manner. A, BMMs were cultured with M-CSF and RANKL for the indicated times. Lysates were immunoblotted with NFATc1, Cbl-b, c-Cbl, c-Src, TRAP, and actin antibodies. B, HEK 293T cells were transfected with FLAG-NFATc1, HA-Cbl-b, and either HA-c-Src-WT or a kinase-dead (KD) mutant form of c-Src (K295M) as indicated. C, HEK 293T cells were transfected with FLAG-NFATc1, HA-c-Cbl, and either c-Src-WT or c-Src-KD as indicated. D, HEK 293T cells were transfected with FLAG-NFATc1, HA-c-Src, and either HA-Cbl-b-WT or a truncation mutant in which the N-terminal SH2 domain of Cbl-b is deleted (ΔN) as indicated. E, HEK 293T cells were transfected with FLAG-NFATc1, c-Src, and either HA-c-Cbl-WT or a ubiquitin ligase-deficient RING finger mutant of c-Cbl (C3AHN) as indicated. F, HEK 293T cells were transfected with FLAG-NFATc1, HA-Cbl-b, and HA-c-Src. MG132 was added 4 h before cell lysis at a concentration of 25 μm as indicated. G, HEK 293T cells were transfected with FLAG-NFATc1, HA-c-Cbl, and c-Src. MG132 was added 8 h before cell lysis at a concentration of 10 μm as indicated. B–G, lysates were immunoblotted with FLAG, HA, and actin antibodies. Results presented are representative of three independent sets of similar experiments.