MOESM2 of Characterization of lysosomal proteins Progranulin and Prosaposin and their interactions in Alzheimer’s disease and aged brains: increased levels correlate with neuropathology

Additional file 2 Figure S2. Western blot characterization of PGRN polypeptides detected with goat anti-PGRN antibody (AF2420). A). Absorption of PGRN polypeptides with peptide-absorbed PGRN antibody. Western blot images of purified PGRN peptide (PGRN), AD brain protein extract (AD) and THP macrophage cell protein extracts (THP) detected with PGRN peptide-absorbed antibody (+Peptide) or non-absorbed antibody (−Peptide). Peptide absorption resulted in almost complete absence of PGRN polypeptide bands. B). Detection of PGRN by western blots is sensitive to reducing agents. Western blots comparing polypeptide bands in protein extracts of samples prepared with DTT (+) or without DTT (−) as reducing agent. Samples: PGRN; purified recombinant PGRN protein. LAN-5; neuronal cells. THP; THP-1 derived macrophages. Brain; AD brain samples. Blots were probed with goat anti-PGRN antibody (R&D Systems, AF2420:50 ng/ml). C). Sensitivity of detection of PGRN polypeptides in brain samples is enhanced by membrane fixation with paraformaldehyde vapors. Western blot images of brain protein samples from low plaque (LP) and Alzheimer’s disease (AD) cases separated under identical conditions without reducing agents. One membrane was fixed with paraformaldehyde vapors (+PFA) compared to membrane not PFA treated (−PFA). Sensitivity of detection is enhanced in PFA fixed membranes. Blots were probed with goat anti-PGRN antibody (R&D Systems, AF2420:50 ng/ml). D). Identification of deglycosylated forms of PGRN. Protein extracts from THP macrophage cells (THP), and LP and AD brain samples were treated with deglycosylation enzyme PNGaseF (+) or control treated (−). Deglycosylation treatment resulted in increased levels of 55 kDa polypeptides and reduced amounts of ~ 75 kDa PGRN band. Blots were probed with goat anti-PGRN antibody (R&D Systems, AF2420:50 ng/ml).