FIGURE 5:

PERK is required for full activation of ATF6 in MEF cells in response to different ER stress conditions. (A) WT MEF cells were treated with 2 μM tunicamycin (TUN) or 1 μM thapsigargin (TG) for between 1 and 6 h, as indicated, or with no stress treatment (0). Equal amounts of the protein lysates prepared from the cultured cells were separated by SDS–PAGE, and the levels of ATF6, ATF4, and actin were measured by immunoblot analysis. The arrows indicate the glycosylated (ATF6-FL/G) and unglycosylated (ATF6-FL/UG) versions of full-length ATF6, and the cleaved ATF6(N) proteins. (B) WT MEF cells were treated with 2 μM tunicamycin (TUN), tunicamycin and 50 μg/ml cycloheximide (TUN/CHX), or cycloheximide alone (CHX) for up to 6 h as indicated. The levels of ATF6-FL/G, ATF6-FL/UG, and actin were measured by immunoblot analysis. (C and D) WT and PERK^−/− MEF cells were treated with tunicamycin (TUN) or thapsigargin (TG) for up to 6 h, and the levels of ATF6, eIF2α∼P, and total eIF2α were measured by immunoblot. The position of the molecular weight standards (kDa) are shown to the right of the ATF6 panel.