Co-immunoprecipitation was performed as previously described (37 (link)–39 (link)), with minor modifications. Briefly, Protein G Beads (15 μL/sample; GE Healthcare, Little Chalfont, United Kingdom) were washed (1× phosphate-buffered saline) and incubated with anti-SMURF1 antibody (5 μg/sample; Abcam, Cambridge, MA) end over end for 4 hours at 4°C. Homogenized NAc tissue (100 μg protein) was incubated overnight at 4°C with 20 μL of bead–antibody complex slurry. Following centrifugation, pellets were washed 3× with 1 mL of wash buffer (1× Tris-buffered saline and 0.1% Triton X-100). After the final wash, pelleted beads were suspended in sample buffer containing sodium dodecyl sulfate and β-mercaptoethanol, heated to 70°C for 10 minutes, and immunoblotted as described above.
Anti smurf1 antibody
Manufactured by Abcam
Anti-SMURF1 antibody is a laboratory research tool used to detect and study the SMURF1 protein. SMURF1 is an E3 ubiquitin ligase involved in the regulation of various cellular processes. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunoprecipitation to analyze SMURF1 expression and localization.
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2 protocols using anti smurf1 antibody
1
Co-immunoprecipitation of SMURF1 in NAc
2
RNA Immunoprecipitation and Target Detection
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RNA immunoprecipitation was performed using the EZMagna RIP kit (Millipore, Billerica, MA, USA) following the manufacturer’s protocol. MIN6 cells were lysed in complete RIP lysis buffer, after which 100 μl of whole-cell extract was incubated with RIP buffer containing magnetic beads conjugated with anti-TRAF3IP2 antibody (Abcam), anti-SMURF1 antibody (Abcam), or anti-Ago2 antibody (CST), negative control normal mouse IgG (Abcam). Samples were incubated with Proteinase K with shaking to digest the protein and then immunoprecipitated RNA was isolated. The RNA concentration was measured using a Microplate reader (Synergy2, BioTek, USA) and the RNA quality assessed using a bioanalyser (Agilent, Santa Clara, CA, USA). Furthermore, purified RNA was subjected to qRT-PCR analysis to demonstrate the presence of the binding targets using respective primers. The primer sequences were listed in Table S6 .
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