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. 2015 Mar 23;10(3):e0119423. doi: 10.1371/journal.pone.0119423

Fig 1. High specificity of Hook isoform-specific antibodies and Hook isoform localization in control brain and AD brain.

Fig 1

(a) Affinity purified Hook isoform-specific antibodies were tested on lysates of N2A cells transfected with pEGFP-Hook plasmids. Lysates of N2A cells expressing EGFP-Hook1 (lane 1), EGFP-Hook2 (lane 2), EGFP-Hook3 (lane 3) and EGFP only (lane 4) were separated on 10% SDS-PAGE and transferred to PVDF membrane. The membrane was probed with 0.5 μg/ml anti-Hook1 antiserum (a-Hk1), 0.5 μg/ml anti-Hook2 antiserum (a-Hk2), 0.5 μg/ml anti-Hook3 antiserum (a-Hk3) or 0.25 μg/ml pan-Hook antiserum (a-pan Hk). Pan-Hook antiserum shows the expression of all EGFP-Hook fusion proteins in N2A cells (100–120 kDa), whereas the isoform-specific antisera only label the corresponding isoform. (b) No crossreactivity of affinity-purified Hook antibodies with PHF-tau prepared from AD brain was detected, phospho-tau antibody AT8 (lane1), a-Hook1 (lane 2), a-Hook2 (lane 3), a-Hook3 (lane 4). (c) The Dot blot shows that Hook2 antibody does not crossreact with β-amyloid; 100 ng EGFP-Hook2 N2A cell lysate (spot 1, 3); 100 ng β-amyloid 1–40 (spot 2, 4). (d) Immunohistochemical staining of the hippocampal CA3 region using Hook1, Hook2 and Hook3 antiserum in control brain and in AD brain (Braak stage V). Hook1 and Hook3 antibodies label tau pathology such as neurofibrillary tangles (asterisk) and dystrophic neurites in neuritic plaques (+). Granular neuronal Hook3 immunoreactivity present in control brains (arrowhead) is reduced in AD and NFT-associated Hook3 immunoreactivity is predominant. Amyloid plaque staining (+) is observed using the Hook2 isoform-specific antibody. Scale bar = 20 μm.