Figure 5.

Interactions with Insig-1-Myc and dislocation of HMG₃₅₀-HA mutants. Cells expressing the indicated constructs of HMG₃₅₀-HA, together (+) or without (−) Insig-1-Myc, were incubated for 2h with ALLN with (+) or without (−) sterols, permeabilized in solution C and fractionated. (A) Aliquots of supernatant and solubilized pellet (10% of samples) were analyzed by SDS-PAGE and IB with the indicated antibodies. (B) The rest 90% of the samples were subjected to IP with an anti-myc monoclonal antibody, and immune complexes were analyzed by SDS-PAGE and immunoblotting (IB) with rabbit anti-myc and anti-HA antibodies for the presence of Insig-1-Myc (ii) and coprecipitated HMG₃₅₀-HA (i), respectively. Closed and open arrowheads indicate glycosylated and un/deglycosylated forms of HMG₃₅₀-HA, respectively. M_r markers (in kilodaltons) are shown.