MOESM5 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 5 Figure S5. Additional analysis and controls for proteins analyzed by co-immunoprecipitation or colocalization with progranulin and prosaposin A-B) Composite western blot of additional potential PGRN-interacting proteins using Protein A or Protein G beads conjugated with normal goat IgG, or antibodies to PGRN, PSAP, sortilin, TMEM106B, cathepsin D and beta secretase-1 (BACE-1) (panel B). Comparison was carried out with non-immunoprecipitated total protein extracts of brain samples analyzed on the same blots. Sortilin: Antibody detected polypeptide with expected molecular weight in brain samples. No sortilin immunoreactive bands were present in PGRN IP samples but a faint band was detected in PSAP IP samples. TMEM106B: Antibody detected polypeptide with expected molecular weight in brain samples. No TMEM106B immunoreactive bands were present in PGRN or PSAP immunoprecipitated samples. Cathepsin D: Antibody detected polypeptide with expected molecular weight in brain samples. No cathepsin D immunoreactive bands were present in PGRN IP samples but a faint band was detected in PSAP IP samples. BACE1: Analysis showed that samples precipitated with PGRN conjugated beads did not pull down BACE1 immunoreactive bands but band detected in PSAP-immunoprecipitated samples. BACE-1 conjugated beads included as positive control. (SH – extract of neuronal SH-SY5Y cells). C) Scatter blot showing ratio of expression levels of PSAP to amounts of PGRN in immunoprecipitated samples. The ratio of PSAP to PGRN present in samples immunoprecipitated with antibody to PGRN was not significantly different between disease groups. Confocal microscopy of PGRN-positive plaque structures with indicated antibodies. D-F) PGRN (green) and Sortilin (red) immunoreactivity was detected in plaques but there was no detectable colocalization (F). Scale bars represent 30 μm. G-I) PGRN (green) and TMEM106B (red) immunoreactivity was detected in or around plaques but there was no detectable colocalization (I). Scale bars represent 30 μm. J-L) PGRN (green) and Cathepsin D (red) immunoreactivity. There was no detectable colocalization (L). Scale bars represent 30 μm. M-O) PGRN (green) and BACE (red) immunoreactivity was detected on cells around plaques but there was no detectable colocalization in cells and on plaques (O). Scale bars represent 30 μm.