Figure 2

Insulin 2 (C96Y)-EGFP is localized to the ER while degradation products appear in the cytosol. A. Clone #4S2 cells were untreated (-Dox) or treated with 2 μg/ml doxycycline (+Dox) for 48 h. Following the treatments, the cells were washed in PBS and lysed in either 1% TX-100 lysis buffer (with protease inhibitors) or directly in SDS sample buffer with β-mercaptoethanol. Equal amounts of protein (or equal volumes) were resolved by SDS-PAGE and immunoblotted using antibodies to GM130 or GFP. B. Cells were untreated (Control) or treated for 48 h with 2 μg/ml doxycycline before lysis and immunoprecipitation with anti-GFP or control mouse IgG antibodies. The entire precipitate (pellets; P) and 10 μg of the supernatant (SN) were resolved by SDS-PAGE and immunoblotted using an anti-GFP (rabbit, polyclonal) antibody. C. Cells were treated with 2 μg/ml doxycycline for 72 h, washed in PBS and homogenized in sucrose buffer as described in the methods. The homogenate was fractionated into membrane (M) and cytosol (C) fractions. D. Cells were treated with 2 μg/ml doxycycline for 72 h, washed in PBS and homogenized in sucrose buffer as described in the methods. The homogenate was fractionated on a linear sucrose density gradient and an equal volume of each fraction was resolved by NuPAGE and immunoblotted using antibodies to HSP90, GRP78, GFP and insulin.