Protein g sepharose

Co-IP study was performed with human primary tissues (n=22). Tissues were lysed as mentioned above (Tissue samples). The lysates were precleared by incubating with protein G Sepharose (GE Healthcare, Uppsala, Sweden) at 4 °C for 2 h. The supernatant was incubated overnight at 4 °C with Noxa, SARM or ubiquitin antibody, followed by incubation of protein G Sepharose for 3 h. The immunocomplexes were washed, resuspended in Laemmli buffer and boiled at 95 °C for 10 min. The co-IP products were immunoblotted and probed sequentially with antibodies to SAG, Noxa, SARM or ubiquitin.

Cells were suspended with IP buffer (25 mM Tris-HCl (pH 7.4), 150 mM KCl, 5 mM EDTA, 5 mM DDT, RNase inhibitor (100 U/ml), protease inhibitor (Roche)) and placed on ice for 15 min, followed by sonication for 2 min. Cell lysates were collected and centrifuged for 10 min at 2,000 rpm at 4°C. Supernatants were preincubated with Protein G Sepharose (GE Healthcare) for 1 h and anti-Ago2 or control IgG at 4°C for 2 h followed by addition of 30 μl of Protein G Sepharose (GE Healthcare) for 1 h. The Sepharose beads were washed three times in PBS and RNAs were extracted using the Qiazol reagent (Qiagen). HCV-RNA associated with Ago2 protein was detected by qRT-PCR as described as above.

Peritoneal macrophages were surface-labeled with biotin (Sigma Aldrich) and lysed in protein sample buffer. Cell lysates were precleared 3 times for 30 min using protein G Sepharose (GE Healthcare) and incubated overnight with anti-m154 mAb and protein G Sepharose. Immunoprecipitates were washed, eluted, subjected to SDS-PAGE in 10% acrylamide gels and transferred to nitrocellulose membranes. Membranes were probed with streptavidin-POD conjugate (Roche Diagnostics GmbH, Mannheim, Germany) and blots developed as for the Western blot analysis.

Recombinant proteins were expressed in HEK-293F (Life Technologies) cells following transient transfection with the Gibco Expi293 expression system kit (Life Technologies). The MST-HN mutations reduce binding of the Fc region to protein G-Sepharose and Seldegs were therefore purified using an anion exchange column (SOURCE-15Q, GE Healthcare) at pH 8.0 and a linear salt gradient (0–0.5 M NaCl). HER2-WT and MOG-WT were purified using protein G-Sepharose (GE Healthcare). 8-18C5 was expressed in recombinant form and purified using protein G-Sepharose23 (link), and clinical grade TZB (Herceptin; Roche) was obtained from the UT Southwestern Medical Center Pharmacy. Recombinant Abdeg (MST-HN, hen egg lysozyme-specific) was purified from culture supernatants using lysozyme-Sepharose1 (link). All recombinant proteins were purified using size-exclusion chromatography (GE Healthcare) in PBS (Lonza) before use in experiments.

After precleaning the extracts with Protein G Sepharose (#17-0618-02, GE Healthcare), samples were subjected to immunoprecipitation with Protein G Sepharose coated with a mouse anti-paxillin antibody. Samples were mixed with an equal volume of 2 × SDS-PAGE sample buffer (containing 200 mM dithiothreitol), boiled for 5 min, and then separated on a 5–20% gradient polyacrylamide gel (#E-R520L, Atto Corp.). Proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore) for 1 h using a conventional semidry electrotransfer (1.3 mA per cm²). The membrane was incubated for 1 h in a blocking solution (For protein detections, 4% nonfat dry milk and 0.1% Tween 20 in TBS, or for pY118-paxillin detection and Tyr-phosphorylation, 1% BSA and 0.1% Tween 20 in TBS) and incubated overnight with indicated primary antibodies, followed by incubation with horseradish peroxidase (HRP)–conjugated secondary antibodies (for rabbit IgG, #RPN4301; for mouse IgG, RPN4201V; GE Healthcare). The binding of these antibodies was detected with Luminata Forte Western HRP substrate (Millipore) and imaged with a chemiluminescent image analyzer system (EZ-Caputure MG, Atto Corp).

48 h after treatment of the MCF7 and MDA-MB231 cells with FBXL8-siRNA or control siRNAs, Co-IP assay was performed. For Co-IP study with MCF7 or MDA-MB231 total cell lysates, the cells were lysed in CHAPS lysis buffer containing 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 137 mM KCl, 1 mM EDTA, 1 mM EGTA, 1% CHAPS and 1 protease inhibitors (complete EDTA-free cocktail, Roche, St. Louis, MO, USA). Then, the cell lysate was precleared by incubating with protein G Sepharose (GE Healthcare, Little Chalfont, UK) at 4 °C for 2 h. The supernatant was incubated overnight at 4 °C with FBXL8 or control IgG_2b antibody, followed by a 3-h incubation with protein G Sepharose. The washed immunoprecipitates, resuspended in Laemmli buffer, was boiled at 95 °C for 5 min before immunodetection of FBXL8, CCND2 or IRF5.