Fig 1.

After prolonged ER stress, degradation of ASGPR H2a cannot be blocked by a proteasomal inhibitor. (A) NIH 3T3 cells stably expressing H2a were preincubated for 16 hours with complete medium in the absence (lanes 1–3) or presence of 10 μg/mL tunicamycin ([tun], lanes 4–6) or 2 μg/mL thapsigargin ([thap], lanes 7–9). They were then metabolically labeled with [³⁵S] Cys and chased for 3 hours with complete medium with or without 20 μM MG-132 as indicated. Tunicamycin or thapsigargin were added also during starvation, pulse, and chase periods. Cell lysates were immunoprecipitated with anti-H2a antibodies and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. On the right are indicated molecular masses in kilodaltons. On the left are the migrations of H2a precursor and cleaved ectodomain fragment. Bands below the upper H2a precursor and fragment species in this and other figures are underglycosylated molecules (with less than the total of 3 sugar chains). This heterogeneity is eliminated in samples preincubated with tunicamycin that blocks N-glycosylation (migration of unglycosylated species is indicated on the left). Cell viability was not very affected by the long preincubation with the unfolded protein response (UPR) inducers, because it did not have a significant effect on the metabolic labeling. (B) RNA was extracted from H2a-expressing cells pretreated with 10 μg/mL tunicamycin for the indicated times and used for reverse transcriptase polymerase chain reaction (RT-PCR) analysis (20 cycles) with primers for BiP mRNA (upper panel) compared to actin (lower panel).These experiments, as well as those in other figures, are representative of at least 3 similar experiments