Detection of DAO and SRR

Copyright information:

Taken from "-Amino acid oxidase and serine racemase in human brain: normal distribution and altered expression in schizophrenia"

The European Journal of Neuroscience 2007;26(6):1657-1669.

Published online Jan 2007

PMCID:PMC2121142.

© The Authors (2007). Journal Compilation © Federation of European Neuroscience Societies and Blackwell Publishing Ltd

(A) Validation of DAO antibody using recombinant flag-DAO expressed in HEK293 cells. Detection with anti-DAO (lanes 1 and 2) and anti-flag (lanes 3 and 4). Flag-DAO is detected as a ∼42-kDa band with anti-DAO (lane 1) and anti-flag (lane 3) in flag-DAO transfected HEK293 cells but not mock transfected cells (lanes 2 and 4). Expressed untagged DAO is detected as a ∼39-kDa band, of predicted size for DAO, with anti-DAO antibody (lane 1 lower band) but not anti-flag (lane 3). (B) Validation of DAO antibody using human tissue samples (Clontech protein medleys); lane 1, cerebellum; lane 2, amygdala; lane 3, hippocampus; lane 4, thalamus; lane 5, spinal cord; lane 6, lung; lane 7, heart; lane 8, kidney. A specific signal for DAO was detected at ∼39 kDa in the cerebellum, spinal cord and kidney. Blots were re-probed with anti-β-actin as a loading control (lower panel). (C) Representative Western blots of DAO (lanes 1 and 2), DAO pre-adsorption control (lanes 3 and 4), and SRR (lanes 5–6) in protein extracts of post-mortem cerebellar tissue from human (lanes 1, 3 and 5) and rat (lanes 2, 4 and 6). DAO was detected at ∼39 kDa and SRR at ∼38 kDa. Blots were simultaneously probed for cyclophilin as a loading control (lower band), which was detected at ∼20 kDa.