Jak1 was ubiquitinated upon RANKL stimulation. (A) BMMs were incubated with 30 ng/mL M-CSF and 100 ng/mL RANKL for the indicated times, and total-cell lysates were subjected to immunoprecipitation using anti-Jak1 antibody. Immunoprecipitates were separated by SDS-PAGE and immunoblotted using antiubiquitin antibody. The blot was stripped and reprobed for Jak1. Jak1 and actin expression was also examined from a small portion of input lysates. (B) BMMs were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 48 hours. MG132 was added for the final 4 hours of incubation. Total-cell lysates were subjected to Western blotting for the examination of Jak1 expression. (A,B) Blots are representative of at least 3 experiments with similar results.