Csde1 immunoblotting

As Csde1 is abundantly expressed in the mouse cortex, cortical tissue homogenates were prepared from mouse brains at different developmental stages. Protein extract of mouse cortical tissue and primary neuronal culture were immersed in radioimmunoprecipitation assay buffer supplemented with a cOmplete protease inhibitor cocktail tablet (Roche, Basel, Switzerland) and sonicated, denatured in 2× SDS loading buffer with 4% β-mercaptoethanol for 10 min at 95°C, and then separated by 12% SDS–polyacrylamide gel electrophoresis. The protein on the gel was transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore, Hessen, Germany), and membranes were blocked with 5% nonfat milk in 1× PBST for 1 hour. The membranes were then incubated in primary antibodies overnight at 4°C, washed three times in 1× PBST, and incubated in secondary antibodies for 1 hour at room temperature. The signals were revealed by HRP reaction using SuperSignal Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA).

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