Fig. 2. N-terminally modified ADPH is resistant to proteolytic degradation. (A) Effects of MG132 concentration on steady-state levels of ADPH variants and TIP47. Representative immunoblots of parental 293 cultures and ADPH variant cultures following a 20-hour incubation in unsupplemented basal culture media containing increasing concentrations of MG132. Anti-β-actin immunoblots are shown beneath the anti-VSV immunoblots. The relative effects of increasing MG132 concentrations on steady-state levels of each ADPH variant and endogenous TIP47 are shown alongside each immunoblot. The values are averages ± s.d. for six samples, each normalized to β-actin. (B) Effects of MG132 and OA on steady-state levels of ADPH[fl]-VSV, 2,3 ADPH-VSV and TIP47. Representative immunoblots of parental 293 cultures and ADPH[fl]-VSV and 2,3 ADPH-VSV cultures following a 20-hour incubation in basal culture media (control), or in basal culture media supplemented with 3 µM MG132 (MG), 300 µM OA, or 3 µM MG132 plus 300 µM OA (MG/OA). The relative effects of these treatments on steady-state levels of each protein are shown alongside each immunoblot. The values are averages ± s.d. for six samples, each normalized to β-actin. (C) Effects of lysosomal protease inhibitors on steady-state levels of ADPH[fl]-VSV, 2,3 ADPH-VSV and endogenousTIP47. Representative immunoblots of parental 293 cultures and ADPH[fl]-VSV and 2,3 ADPH-VSV cultures after a 20-hour incubation with the indicated inhibitors of lysosomal proteases. The relative effects of these inhibitors on steady-state cellular levels of each protein are shown alongside each blot. The values are averages ± s.d. for six samples, each normalized to β-actin. All experiments were repeated twice, with similar results. In-depth statistical analyses of these results are given in the text.