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. 2009 Feb 16;206(2):287–298. doi: 10.1084/jem.20081335

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© 2009 Nice et al.

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Figure 5. — Mult1 induction by UV irradiation is accompanied by a decrease in ubiquitination. (A) Fibroblasts transduced with FLAG-tagged Mult1 were exposed to 50 J/M² UV-C irradiation. After UV treatment for 4 or 8 h, cells were stained with FLAG antibody and analyzed by flow cytometry. Cells treated in duplicate were used for Mult1 quantitative PCR and normalized to GAPDH. Data shown is mean ± range; n = 2. (B) Untransduced fibroblasts were UV irradiated as in Fig. 1 A, followed by staining with Mult1 antibody at the indicated time after UV treatment. Cells treated in duplicate were used for Mult1 quantitative PCR as in A. (C) B6 fibroblasts transduced with H60a or the H60a-Mult1 chimera (the H60a extracellular domain with the TM and IC domains of Mult1) were UV irradiated as in Fig. 1 A, followed by staining with Mult1 or H60a antibody at 6 h after treatment. Parallel cultures of cells transduced with the H60a-Mult1 chimera were used for semiquantitative RT-PCR to quantify transcripts of the chimeric protein, endogenous Mult1, and HPRT. Solid gray histograms and dashed gray lines in A–C represent isotype staining of untreated and treated cells respectively. (D) Fibroblasts transduced with indicated retroviral construct were treated with 50 J/M² UV-C irradiation and scraped into RIPA buffer 6 h after UV treatment. Cleared lysates were immunoprecipitated with Mult1 antibody and Western blotted with FLAG or ubiquitin antibodies. Vec., Vector transduced; WT, WT Mult1 transduced; KR, Lys to Arg mutant Mult1 transduced cells. Data in this figure is representative of at least three independent experiments.