Anti myc

To test cellular interaction of HOIP with IpaH1.4/2.5, Cys to Ser mutations in IpaH proteins (IpaH1.4C368S, IpaH2.5C368S, IpaH4.5C379S, IpaH7.8C357S and IpaH9.8C337S) were generated by Quickchange mutagenesis (Stratagene). GFP-IpaHC/S proteins were co-expressed with Myc- or Flag-tagged HOIP in 293T cells. 38 h after transfection cells were collected, washed once in cold PBS and lysed in buffer containing 25 mM Tris-HCl (pH7.4), 150 mM NaCl, 1% Triton, 1x protease inhibitor (Sigma Aldrich). Lysate was first pre-cleared with 15 μl ProteinA agarose beads (Thermo Scientific) for 1h. To IP Myc-HOIP or Flag-HOIP 2 μg anti-Myc (Covance) or anti-Flag (Sigma Aldrich) antibody was added for 1h followed by addition of 15 μl Protein-A agarose beads for 1.5h. Beads were then washed 10 times in lysis buffer and 50 μl 2x SDS loading buffer (Biorad) was added. For mapping experiments IP was performed using 2 μg anti-GFP antibodies (Clontech).

HEK 293T cells were lysed in NP-40 buffer and then were centrifuged at 15,890g for 20 min. To detect the polyubiquitylation of TRIM25, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer [50 mM tris-HCl (pH 7.4), 150 mM NaCl, 1% (v/v) NP-40, 0.5% deoxycholate, 0.1% SDS, and protease inhibitor cocktail]. For coimmunoprecipitations, 1 ml of postcentrifuged lysates was incubated with 0.5 to 2.5 μg of antibody at 4°C overnight, followed by incubation with a 50% slurry of protein A/G agarose (Amersham) for 2 hours at 4°C. Immunoprecipitated proteins were extensively washed with lysis buffer and were eluted with SDS–Laemmli buffer by boiling for 5 min. For Western blotting analysis, proteins were resolved by SDS-PAGE and were transferred onto polyvinylidene difluoride membranes. The following primary antibodies were used at 1:2000 dilution: anti-V5 (Invitrogen), anti-FLAG (M2; Sigma), anti-Myc (Covance), anti-GST (Sigma), anti-HA (HA-7; Sigma), monoclonal anti-TRIM25 (BD Biosciences), and anti–β-actin (Abcam). The following antibodies were used at 1:500 dilution: anti-USP15 (1C10; Abnova), anti-USP11 [GeneTex (clone EPR4346)], and anti-ubiquitin (P4D1; Santa Cruz Biotechnology). Protein bands were visualized with the enhanced chemiluminescence reagent (Pierce) and were detected by a luminescent imaging system (Fuji LAS-4000).