Cathepsin D (CathD) and B (CathB) expression and localization in primary neurons. A. Western blot analyses of CathD and CathB protein levels after transfection. Primary cortical neurons were transfected with 23QHtt, 23QHtt plus CathD, 23QHtt plus CathB, 145QmHtt, 145QmHtt plus CathD and 145QmHtt plus CathB constructs. Western blot analyses were performed with anti-CathD and anti-CathB antibodies. β-actin western blots were used as loading controls. Relative expression levels were quantified by band intensity. Positions of molecular weight markers were indicated. Quantification of the western blots are shown in the bar graphs. *p < 0.05 compared to without cathepsin transfection. B. Analyses of CathD and CathB enzymatic activities after transfection. Primary cortical neurons were transfected with CathD, CathB and as described in A. CathD and CathB activities were assayed by CathD or CathB activity assay kit. *p < 0.05 compared to without cathepsin transfection. C and D. Colocalization of transfected CathD (C) and CathB (D) with LAMP-1. CathD and CathB transfected neurons were examined by co-immunostaining of LAMP-1 and CathD or CathB. Yellow colored cytoplasmic spots are indicative of co-localization of transfected cathepsins and LAMP-1. Scale bar = 10 micron.