Quantitative Western Blot Analysis of Rat Brain Proteins

Western blotting analysis was performed as previously reported [28 (link)]. Whole-cell lysates were used to extract proteins from rat brain tissues in radioimmunoprecipitation assay (RIPA) lysis buffer and were quantified for protein concentration using a bicinchoninic acid (BCA) kit. The protein sample was mixed with the loading buffer and heated in a boiling water bath for denaturation. The same concentration of denatured protein sample was added to each loading well. Proteins were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes using a semidry transfer system (Bio–Rad, USA). After blocking in defatted milk powder for 1 h at room temperature, the membranes were washed with phosphate buffer solution with Tween 20 (PBST) and incubated overnight at 4°C with primary antibodies against FBXW7, HIF-1α VEGF and GAPDH (all purchased from Abcam). Next, the membranes were washed with PBST (5 times × 3 min) and incubated with HRP-secondary antibodies for 1 h. Blots of target proteins were developed using an enhanced chemiluminescence (ECL) kit. Using GAPDH as the loading control, the relative expression of target proteins is expressed as the gray value ratio of the target protein to GAPDH.