Polyvinylidene di uoride membranes

We detected the amounts of each protein in the cell lysates as previously described [11, 12, 13] . The cells were lysed with homogenization buffer (20 mM Tris-HCl (pH 8.0) containing 137 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 10 µg/ml leupeptin and 10 µg/ml pepstatin A). After the protein concentration was determined using Bradford Protein Assay Dye Reagent (Bio-Rad), each cell lysate was dissolved in an equal amount of 2⋅ sodium dodecyl sulfate (SDS)-Laemmli sample buffer (62.5 mM Tris-HCl (pH 6.8) containing 2% SDS and 10% glycerol), and equal amounts of cell lysate were prepared. Equal amounts of protein were separated on 10 or 12.5% SDS-polyacrylamide gels, transferred onto polyvinylidene di uoride membranes (GE Healthcare) and identi ed by enhanced chemiluminescence (GE Healthcare) using antibodies against CREB3 (Proteintech and Santa Cruz), CREBRF (Abcam), EGFP (Roche), a myc-epitope and CREB3 (Santa Cruz Biotechnology) and G3PDH (Acris Antibodies). The expression level of each protein was analyzed using ImageJ software (National Institutes of Health), and the relative amount of cleaved CREB3 was calculated based on the G3PDH value obtained from the same lysate. The cleaved CREB3 protein expression levels of each lysate were normalized to the value obtained from the BFA-treated Neuro2a cells [13] .