Chemidoc xrs system image analysis software

Cells were transfected with equimolar concentrations of TBR1 expression plasmids. Whole-cell lysates were extracted by treatment with lysis buffer (100 mM Tris pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.2% Triton X-100, 1% PMSF, 1 × protease inhibitor cocktail; all from Sigma) for 10 min at 4 °C, before centrifuging at 10,000 g for 30 min at 4 °C to remove cell debris. Proteins were resolved on 4–15% Tris–Glycine gels and transferred onto polyvinylidene fluoride membranes. Membranes were probed with Invitrogen mouse anti-Xpress (for pcDNA4.HisMax constructs; 1:1,000) or Clontech mouse anti-EGFP (for pYFP constructs; 1:8,000) overnight at 4 °C, followed by incubation with HRP-conjugated goat anti-mouse IgG for 45 min at room temperature (Bio-Rad; 1:2,000). Proteins were visualized using Novex ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) and the ChemiDoc XRS +System (Bio-Rad). Equal protein loading was confirmed by stripping blots and reprobing using Sigma anti-β-actin antibody (1:10,000). Bands were quantified by densitometry using the ChemiDoc XRS +System image analysis software (Bio-Rad). A value for the transfection efficiency of TBR1 constructs was obtained by dividing the YFP signal by the β-actin signal. The relative expression of TBR1 variants was then derived by dividing the Xpress signal by the transfection efficiency.