Co-IP experiments were performed in low-light conditions [see sample guidelines for working with S-nitrosylation (41)]. For neuronal co-IPs, cells were seeded onto 10 cm plates, treated with 200 μM CysNO donor or Cys control for 20 min, then washed with cold PBS, and harvested in cold IP lysis buffer [50 mM tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, and 1% sodium deoxycholate (DOC)] containing a protease inhibitor cocktail, phosphatase inhibitors 2 and 3, and phenylmethylsulfonyl fluoride (all at 1:100). Samples were lysed on ice for 30 min, homogenized, and then cleared by centrifugation at 1000g for 10 min at 4°C. Lysates were precleared with protein G Sepharose beads (catalog no. 17-0618-01, GE Healthcare) for 2 hours at 4°C. Total inputs (10%) were taken. The remaining sample was incubated overnight 4°C with rabbit anti-CHD4 (ab72418) or rabbit IgG (X0903, Dako). Lysates were then incubated with protein G Sepharose beads for 2 hours at 4°C. Beads were washed once with wash buffer [50 mM tris (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, 5% glycerol], with protease, and phosphatase inhibitors at 1:1000 twice with wash buffer, and twice with PBS. Excess PBS was removed, and proteins were eluted by boiling the beads with 2× Western loading buffer. Total inputs and samples were then run on precast 3 to 8% tris-acetate gels.

For HEK293T cell co-IPs, cells were seeded onto 6 cm plates and cotransfected with Flag-tagged mCHD4 and either HA-RBBP7WT or HA-RBBP7C166S. Cells were then treated as described for E17 neurons, then washed with cold PBS, and harvested in cold radioimmunoprecipitation assay buffer [50 mM tris (pH 7.5), 150 mM NaCl, 1% NP40, and 0.5% DOC] containing inhibitors as described above at 1:100. Samples were lysed and cleared, and then, protein concentration was determined using BCA Protein Assay; 250 to 400 μg of protein was used per experiment. Lysates were precleared, and total inputs were taken. The remaining sample was incubated overnight in the dark at 4°C with rabbit anti-HA (3724S, Cell Signaling Technology) or rabbit IgG (X0903, Dako). Lysates were then incubated with protein G–Sepharose beads for 2 hours at 4°C. Beads were washed, and proteins were eluted as for E17 neuron samples. Samples were then run on precast NuPAGE 4-12% bis-tris gels.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.