FIGURE 4.

Upon proteasomal inhibition H2aΔgly colocalizes with EDEM1 and ERAD factors at the ERQC. A, plasmids encoding for H2a-RFP and myc-tagged H2aΔgly were cotransfected in NIH 3T3 cells. One day after transfection, cells were incubated for 3 h without (upper panels) or with Lac (25 μm) (lower panels), fixed, permeabilized, and incubated with mouse anti-Myc and FITC-conjugated goat anti-mouse IgG. Representative confocal optical slices are shown. B, similar to A but with endogenous GM130 detected with rabbit anti-GM130 and Cy3-conjugated goat anti-rabbit IgG. C, cells cotransfected with HA-tagged EDEM1 and untagged H2aΔgly and incubated with rabbit anti-H2a and Cy3-conjugated goat anti-rabbit IgG and with mouse anti-HA and FITC-conjugated goat anti-mouse IgG. D, similar to B; endogenous Derlin-1 was visualized using rabbit anti-Derlin-1 and Cy3-conjugated goat anti-rabbit IgG. E, similar to C but with Myc-tagged HRD1 instead of EDEM1-HA. Mouse anti-Myc antibodies and FITC-conjugated goat anti-mouse IgG were used to visualize HRD1. Bar, 10 μm. F, similar to C with cells expressing H2aΔgly and FLAG-tagged Fbs2, detected with mouse anti-FLAG and FITC-conjugated goat-anti mouse IgG. G, cells transfected with Myc-tagged H2aΔgly and GalT-YFP, incubated with mouse anti-Myc and goat-anti mouse Dylight549 IgG. Endogenous Bip was visualized using rabbit anti-Bip and goat anti-rabbit DyLight650 IgG, pseudocolored blue. A section was enlarged for better detail of the lack of colocalization.