Figure 1

Cathepsin D (CathD) and B (CathB) reduce Htt loads in HEK cells. HEK cells transfected by lipofectamine with different Htt and cathepsin cDNA constructs were harvested 48 hr later for real-time quantitative RT-PCR, western blot and enzymatic activity analyses. A. Transfection of cathepsin D (CathD) and B (CathB) increases their expression in HEK cells. HEK cells were transfected with 23QHtt, 23QHtt plus CathD, 23QHtt plus CathB, 145QmHtt, 145QmHtt plus CathD, and 145QmHtt plus CathB constructs for 48 hr. Western blot analyses were performed with anti-CathD and anti-CathB antibodies. β-actin western blots were used as loading controls. Pre-pro forms of CathD are 49 and 50 kD. Mature CathD is 32 kD. Mature CathB is 27 kD. Relative expression levels were quantified by band intensity. Student t-test was performed. *p < 0.05 compared to without cathepsin transfection. B. Increase of CathD and CathB enzymatic activities after transfection. HEK cells were transfected with CathD and 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-E) Transfection of CathD and CathB reduce transfected Htt levels. HEK cells were transfected with 23QHtt, 23QHtt plus CathD, 23QHtt plus CathB, 145QmHtt, 145QmHtt plus CathD, and 145QmHtt plus CathB constructs. Western blot analyses were performed first with (C) 1C2 antibody that is specific for the polyQ of 145QmHtt, and then with (D) EM48 that preferentially recognizes the aggregates or (E) Ab2166 antibody that recognizes both 23Q and 145QHtt. β-actin western blots were used as loading controls. *p < 0.05 compared to without cathepsin transfection. †p < 0.05 compared to 23Q transfection.