Journal:

Article Title: E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection

doi: 10.1016/j.virol.2008.08.045

Figure Lengend Snippet: The E4 34k/ligase IV complex observed in extracts from H5dl1016-infected cells contained Elongins C and B. HeLa cells were Mock-, Ad5- or H5dl1016-infected and lysed in NP40 lysis buffer 20 h post-infection. (A) 200 μg of extract was subject to IP with antibodies directed against Elongin B (top) or Elongin C (bottom). Elongin-containing complexes were resolved by SDS-PAGE, Western transferred and probed for E4 34k. (B) Complexes containing Elongin B (top) and C (bottom) were isolated as described in (A), resolved by SDS-PAGE, Western transferred and probed for Ligase IV. I, 15% of input lysate; IP, immunoprecipitation using anti-Elongin antibodies; C, control IP. M, A and 16; control IP carried out with lysates from Mock-, Ad5- or H5dl1016-infected cells, respectively. IP: Elongin B panel was assembled from lanes from a single Western blot.

Article Snippet: Elongin C IPs: anti-Elongin C mAb (BD transduction laboratories), 1:10 dilution.

Techniques: Infection, Lysis, SDS Page, Western Blot, Isolation, Immunoprecipitation

Journal:

Article Title: E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection

doi: 10.1016/j.virol.2008.08.045

Figure Lengend Snippet: Expression of E4 34k alone is not sufficient to disrupt the ligase IV/XRCC4 complex in human cells. (A) Expression of Ad5 late proteins in H5dl1011-infected RKO-E4 cells. RKO and RKO-E4 cells were mock-(M), Ad5-, or H5dl1011-infected (1011). Extracts were prepared 24 or 48 h post-infection (HPI), resolved by SDS-PAGE, Western transferred and probed for adenoviral late proteins, E4 34k and XRCC4. Top panel showing late protein expression was assembled from lanes from a single Western blot. (B) Control RKO and E4 34k-expressing RKO-E4 cells were lysed in NP40 lysis buffer and 200 μgof extract was subject to IP with antibodies directed against XRCC4. Immune complexes were resolved by SDS-PAGE, Western transferred and probed for Ligase IV, XRCC4 and Actin. I, 7.5% of input lysate; IP, immunoprecipitation using anti-XRCC4 antibodies; C, control IPs. (C) RKO and RKO-E4 cells were lysed in NP40 lysis buffer and 200 μgof extract was subject to IP with antibodies directed against E4 34k. Immune complexes were resolved by SDS-PAGE, Western transferred and probed for E4 34k, Ligase IV, Elongin B, Elongin C and Actin. I, 10% of input lysate; IP, immunoprecipitation using anti-E4 34k antibodies; C, control IPs.

Article Snippet: Elongin C IPs: anti-Elongin C mAb (BD transduction laboratories), 1:10 dilution.

Techniques: Expressing, Infection, SDS Page, Western Blot, Lysis, Immunoprecipitation

Journal:

Article Title: E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection

doi: 10.1016/j.virol.2008.08.045

Figure Lengend Snippet: Schematic representation of E4 34k-mediated ligase IV recognition in adenovirus-infected cells. (A) In cells infected with adenovirus expressing E4 34k, but not E1B 55k, observation of an E4 34k/Cul5/Elongin BC/ligase IV complex and coincident observation of XRCC4/DNA-PKcs complexes that lack ligase IV indicate that assembly of the E4 34k/Cul5/Elongin BC ubiquitin E3 ligase and binding to ligase IV results in dissociation of the strong, functional XRCC4-ligase IV direct interaction. Rbx1 (white) is predicted to assemble with the E4 34k/Cul5/Elongin BC ubiquitin E3 ligase. (B) Model predicts that in the cells infected with adenovirus expressing both E4 34k and E1B 55k, assembly of the E4 34k/E1B 55k/Cul5/Rbx1/Elongin BC ubiquitin E3 ligase and binding to ligase IV results in ligase IV/XRCC4 dissociation. Polyubiquitination of ligase IV and proteasome-mediated degradation of ligase IV follows.

Article Snippet: Elongin C IPs: anti-Elongin C mAb (BD transduction laboratories), 1:10 dilution.

Techniques: Infection, Expressing, Binding Assay, Functional Assay