LMP1 and TRAF1 complexes are modified by M1-pUb chains.

<p>A) FLAG-LMP1 complexes were immuno-purified from LCLs that express FLAG-LMP1 from the EBV genome at physiologic levels [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004890#ppat.1004890.ref018" target="_blank">18</a>]. GM12878 LCLs were used as a negative control. M1-linked polyubiquitin chain (M1-pUb) content was analyzed by WB, using a highly chain specific antibody. * indicates antibody heavy chain background. FLAG-LMP1 migrates at a slightly higher than untagged LMP1. B) FLAG complexes were immuno-purified from GM12878 LCLs that stably express either FLAG- GFP or FLAG-TRAF1, and were immuno-blotted, as indicated. C) 293 cells were co-transfected with FLAG- GFP, FLAG-TRAF1, or FLAG-TRAF2, and either empty PSG5-vector control or untagged LMP1 1–386 for 24 hours. Purified FLAG complexes or whole cell lysates were blotted for FLAG or LMP1, as indicated. D) FLAG-LMP1 LCLs were transduced with lentiviruses that express control shGFP or an independent shRNA against HOIP. Following puromycin selection, FLAG LMP1-immuno-purified complexes and lysates were blotted, as indicated. E) Whole cell extracts from 293 TRAF1 cells, uninduced or induced for LMP1 1–231 expression for 16 hours, were blotted for M1-pUb, LMP1, or tubulin. A-E are representative of three independent experiments.