Phospho creb antibody

Cells were lysed in RIPA buffer and centrifuged at 13,500 g for 10 min at 4°C. Supernatants were measured with a Bradford assay (Bio-Rad Laboratories, Inc.) and 30 µg of total protein were separated using a 10% SDS-PAGE gel and then transferred to a polyvinylidene difluoride membrane. The membrane was blocked in 5% nonfat dried milk in TBST (TBS containing 0.1% Tween-20) for 1 h at room temperature, followed by immunoblotting and incubation overnight at 4°C with the primary antibodies as follows: CREB (1:1,000, Cell Signaling Technology, cat. no. #4820), phospho-CREB antibody (1:1,000; Cell Signaling Technology, #9198), Ngb (1:500; Abnova, 2B5-A7), β-actin antibody (1:3,000; Sigma, A5441). Subsequently, the membrane was incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (both 1:5,000; cat. nos. #31430 and #31460; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. After washing with TBST, immunoreactive proteins were determined by an enhanced chemiluminescence substrate (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Images were captured with ChemiDoc Imaging Systems (Bio-Rad Laboratories). Quantification of protein band intensity obtained by Western blotting analysis was analyzed with ImageJ 1.52a (http://rsb.info.nih.gov/ij/) and normalized to the actin band intensity.