Ubiquitination is involved in the inhibition of Mult1 expression. (A) Fibroblasts transduced with FLAG-tagged Mult1 were lysed in RIPA buffer and Mult1 was immunoprecipitated with Mult1 or control antibody, followed by Western blotting with either ubiquitin or FLAG antibodies. Iso., isotype control; Vec., empty vector transduced; WT, WT Mult1 transduced; KR, Lys to Arg mutant Mult1 transduced. (B) Untransduced, WT Mult1–transduced, or KR Mult1–transduced fibroblasts were treated with either DMSO, 20 µM MG132, or 100 µM chloroquine for 6 h, followed by staining with FLAG antibody and analysis by flow cytometry. (C) Untransduced, WT Mult1–transduced, or KR Mult1–transduced fibroblasts were grown on tissue culture–treated glass slides and treated with DMSO, 20 µM MG132, or 100 µM chloroquine for 6 h. Cells were permeabilized, stained with Mult1 antibodies followed by Alexa Fluor 555–conjugated secondary antibodies and DAPI, and analyzed by immunofluorescence microscopy. Bar, 10 µm. Data in this figure is representative of at least four independent experiments.