Figure 5.

Like CNX, upon proteasomal inhibition H2a is transported to the ERQC. CNX relocalization is independent of the substrates. (A–F) Similar to Figure 1 but with NIH 3T3 cells stably expressing H2a incubated for 5 h with or without 10 μM Lac as indicated. The antibodies used were rabbit polyclonal anti-H2 carboxy-terminal with Cy3-conjugated goat anti-rabbit IgG and mouse monoclonal anti-β-COP with FITC-conjugated goat anti-mouse IgG. (G–I) The same cells as in A–F were incubated with 10 μg/ml tunicamycin for 3 h after which 10 μM Lac was added for an additional 3 h. Cells were then fixed and reacted with rabbit polyclonal anti-CNX and Cy3-conjugated secondary and mouse monoclonal anti-β-COP with FITC-conjugated secondary. (J–U) NIH 3T3 cells were transiently transfected with a plasmid carrying H2a-dsRed, and after 48 h they were incubated for 3 h with or without 10 μM Lac, fixed, and reacted with rabbit anti-CNX or anti-ERp57 antibodies as indicated using FITC-conjugated goat anti-rabbit IgG as secondary. Bars, 10 μm.