Protein affinity purification, coimmunoprecipitation, and Western blot analyses were performed as previously described (29, 30). For coimmunoprecipitation experiments, 500 to 700 μg of solubilized protein extracted from mouse brain tissue or HEK293T cells (American Type Culture Collection) was incubated in the presence of primary antibodies or immunoglobulin G (negative control) (1 to 2 μg) together with protein A/G plus agarose (25 μl per sample; catalog number: sc-2003, Santa Cruz Biotechnology) with gentle shaking for 12 hours at 4°C. Pellets were washed, boiled for 5 min in SDS sample buffer (catalog number: 161–0737, Bio-Rad) and 2-Mercaptoethanol (M7154, Sigma-Aldrich), and subjected to SDS–polyacrylamide gel electrophoresis (PAGE). A total of 50 to 100 μg of protein extracted from tissue was used as a control in each experiment. For protein affinity purification experiments, 500 to 700 μg of protein was incubated with Glutathione Sepharose 4B (30 μl per sample; catalog number: 27-4574-01, Amersham) bound to the indicated GST fusion proteins (50 to 100 μg) at 4°C overnight. Beads were washed, boiled for 5 min in SDS sample buffer, and subjected to SDS-PAGE. After transfer of proteins onto nitrocellulose, membranes were Western-blotted with the primary antibodies specified in the antibody information. The intensity of protein abundance was quantified by densitometry (software: ImageJ from National Institutes of Health and Image Lab from Bio-Rad).

The antibodies used were those against D1R (2 μg per sample; goat, catalog number: sc-1434, Santa Cruz Biotechnology), SynGAP (2 μg per sample; rabbit, catalog number: sc-33598, Santa Cruz Biotechnology), and GFP (2 μg per sample; rabbit, catalog number: sc-8334, Santa Cruz Biotechnology) for immunoprecipitation; D1R (1:1000; rat, catalog number: D2944, Sigma-Aldrich), SynGAP (1:500; rabbit, catalog number: sc-33598, Santa Cruz Biotechnology), and GFP (1:1000; mouse, catalog number: sc-9996, Santa Cruz Biotechnology) for Western blotting; phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000; rabbit, catalog number: 9101, Cell Signaling Technology); p44/42 MAPK (ERK1/2) (1:1000; rabbit, catalog number: 9102, Cell Signaling Technology); PKA C-α (1:1000; rabbit, catalog number: 5842, Cell Signaling Technology); phospho PKA α+β (catalytic subunits) (Thr197) (1:1000; rabbit, catalog number: ab5815, Abcam); p38 MAPK (1:1000; rabbit, catalog number: 9212, Cell Signaling Technology); phospho–p38 MAPK (Thr180/Tyr182) (1:1000; rabbit, catalog number: 4511, Cell Signaling Technology); pan-phosphorylation (1:1000; rabbit, catalog number: 61-8300, Invitrogen); MAP2 (1:500; rabbit, catalog number: AB5622, Millipore); and cortactin (1:1000; mouse, catalog number: ab33333, Abcam).

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

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.