Cell total protein extraction kits

Total protein was prepared using the cell total protein extraction kits according to the manufacturer’s instruction (Millipore, Billerica, MA). Equal concentrations of each protein sample (20 μg) were separated on 6 % SDS polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon P, Millipore, Bedford, MA, USA). The membranes were blocked with 5 % nonfat milk in Tris-buffered saline containing 0.1 % Tween 20 (TBST) for 1 h at room temperature, and incubated a primary monoclonal antibody to CEP55 (1:1000, Abcam, USA, ab170414) overnight at 4 °C. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA, SC-2004). The ECL prime Western blotting detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ) was used to detect CEP55 expression according to the manufacturer’s instructions. GADPH (Santa Cruz Biotechnology) was used to confirm equal loading of the samples.

Total protein was prepared using the cell total protein extraction kits according to the manufacturer’s instruction (Millipore, Bedford, MA). Equal amounts of each protein extract (30 μg) were electrophoretically separated on 9.5% SDS polyacrylamide gels. Following transfer to polyvinylidene fluoride (PVDF) membranes (Immobilon P, Millipore, Bedford, MA), membranes were blocked for 1 h at room temperature with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). The membranes were then incubated with anti-B3GNT3 antibody (1:1000, Proteintech, 18098-1-AP) overnight at 4°C. Horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:2000, Santa Cruz, SC-2004) was used to determine B3GNT3 expression. Finally, B3GNT3 expression was detected using ECL prime Western blotting detection reagent (Amersham) according to the manufacturer’s instructions. Blotted membranes were stripped and re-probed with an anti-Alpha-Tubulin antibody (Sigma, Saint Louis, MO) as a loading control.