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. 2017 Jul 27;13(7):e1006542. doi: 10.1371/journal.ppat.1006542

Fig 7. IE1 induces ubiquitination of Sp100A and assembles an Sp100A ubiquitination complex.

Fig 7

(A-B) Effect of IE1 on the protein levels of endogenous and exogenous Sp100A in 293T cells. 293T cells were transfected with pEYFP-IE1 (IE1) or pEYFP (vector) in the absence (A) or presence (B) of pCMV-Myc-Sp100A (Myc-Sp100A). Cells were harvested at 48hpt for immunoblotting. Endogenous (A) or exogenous (B) Sp100A levels were examined by antibodies against Sp100A or Myc, respectively. The values listed below the blots indicate the relative protein levels of Sp100A compared to the controls following β-actin normalization. Representative images of IB from 3 independent experiments are shown (upper panel), and relative levels of Sp100A are presented as the mean ± SD (lower panel). **, P≤0.01. (C-D) Effect of IE1 on ubiquitination of exogenous Sp100A in 293T cells. 293T cells were transfected with 0.1μg pCMV-Myc-Sp100A (Myc-Sp100A) and 2μg pEYFP-IE1 (IE1), pEYFP-IE1(Δ451–475) (Δ451–475), or pEYFP (vector). At 36hpt, cells were treated with MG132 (+) or DMSO (-) for 12 h. Cell lysates were subjected to Myc-directed IP and subsequent IB for ubiquitin (Ub) and Myc. The indicated proteins were also examined in the cell lysates. (E) Effect of IE1 on assembling the Sp100A ubiquitination complex. Purified His-Sp100A was mixed with Ubc5a in the presence or absence of purified His-IE1. The mixtures were then subjected to Sp100A- or Ubc5a-directed IP, followed by IB for the indicated proteins. Representative results from 3 independent experiments are shown. Normal IgG was used as the nonspecific antibody control. The input components in the reaction were examined, and the indicated proteins are shown. (F) Effect of IE1 on ubiquitination of Sp100A in vitro. Purified His-IE1 or His-Δ451–475 were mixed together with His-Sp100A, E1, E2 (Ubc5a) and ubiquitin as described above and in Materials and Methods, and incubated at the indicated temperature for 2h to perform in vitro ubiquitination reaction. The mixtures were subjected to Sp100A-directed IP and subsequent IB for ubiquitin (Ub). The input components in the reaction were examined, and the indicated proteins are shown. The mono-ubiquitinated Sp100A is indicated by an asterisk (*) according to the protein size.