Fig 1: Effect of purified EDEM1/2/3 on M8B present in ATF6a(C).(A) (a) Eluate obtained from EDEM1, 3-double knockout (DKO) cells overexpressing ATF6a(C)-TAP2 was subjected to SDS-PAGE under reducing conditions, silver-stained, and analyzed by immunoblotting using anti-Myc antibody. (b) Eluate in (a) was untreated (-) or treated (+) with EndoH, subjected to SDS-PAGE under reducing conditions, and analyzed by immunoblotting using anti-Myc and anti-GRP78 (which is identical to BiP) antibodies. (B) (a) Eluate in (A) was incubated with purified EDEM1, EDEM2-TXNDC11 complex, or EDEM3(Q543N) for the indicated time, and then analyzed by immunoblotting using anti-Myc antibody. (b) N-glycans prepared from samples in (a) after 24 hr incubation were analyzed by mass spectrometry (MS). This experiment was conducted once.
Fig 2: Effects of IDN5706 on APP turnover at the ER.(A) Similar to unfolded luminal glycoproteins that are recruited by EDEM1 from the Calnexin/Calreticulum (CNX/CLR) folding cycle (1), a fraction of newly-synthesized, immature APP (iAPP) is substrate of the endoplasmic reticulum-associated protein degradation (ERAD) (2). (B) The levels of EDEM1 are reduced upon IDN5706 treatment by a process independent on the levels of Atg5. This is a mechanism that needs further elucidation (?). Accumulation of iAPP in the ER elicited by IDN5706 activates an alternative early degradation of iAPP by Atg5-dependent autophagy (3).
Fig 3: Effect of four purified a1,2-mannosidases on PA-M8B.(A) Wild-type (WT) cells were transfected with plasmid to express mCD3-d-?TM-HA. EDEM2-knockout (KO) cells and EDEM1, 3-double knockout (DKO) cells were transfected with plasmid to express mCD3-d-?TM-HA together with or without plasmid to express TAP-EDEM1, TAP-EDEM2, TAP-EDEM3(Q543N), or TAP-MAN1B1(?105). Cell lysates were then prepared and analyzed by immunoblotting using anti-HA and anti-Myc antibodies. (B) Eluates obtained from WT cells untransfected (-) or transfected with plasmid to express TAP-EDEM1, TAP-EDEM2 plus TXNDC11(M1A), EDEM3(Q543N), or MAN1B1(?105) were subjected to SDS-PAGE under reducing conditions, silver-stained, and then analyzed by immunoblotting using anti-Myc antibody. PA-M8B was incubated with samples in (B) for 24 hr as indicated and then analyzed by HPLC (amide column) for mannose contents. (D) The M7 peak (a), M6 peak (b), and M5 peak (c) obtained in (C) were analyzed by HPLC (ODS column) for isomer identification. (E) Route of oligosaccharide processing in the mammalian endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD) is shown.
Fig 4: Effect of purified EDEM1/2/3 on M8B present in mCD3-d-?TM-HA.(A) (a) Eluate obtained from EDEM1, 3-double knockout (DKO) cells overexpressing TAP-mCD3-d-?TM-HA was subjected to SDS-PAGE under reducing conditions, silver-stained, and then analyzed by immunoblotting using anti-HA antibody. (b) Eluate in (a) was untreated (-) or treated (+) with EndoH, subjected to SDS-PAGE under reducing conditions, and analyzed by immunoblotting using anti-HA, anti-Myc, and anti-GRP78 (which is identical to BiP) antibodies. (B) (a) Eluate in (A) was incubated with purified EDEM1, EDEM2-TXNDC11 complex, or EDEM3(Q543N) for the indicated time, and then analyzed by immunoblotting using anti-Myc antibody. (b) N-glycans prepared from samples in (a) after 24 hr incubation were analyzed by mass spectrometry (MS). This experiment was conducted once.
Fig 5: IDN5706 reduces the levels of EDEM1 in a time-dependent manner.(A) H4 cells were left untreated or treated for different periods of time with 250 µM IDN5706, and subsequently analyzed by Western blotting with an EDEM1 antibody. The antibody specificity was validated on protein extracts from cells stably expressing either luciferase shRNA, used as positive control (shLuc), or EDEM1 shRNA (shEDEM1). Specific antibodies against ER proteins including Calnexin, Gp78 and Bip were tested. (B) Densitometric quantification of the levels of EDEM1 in cells left untreated or treated with IDN5706 for 16 h, as shown in A. Bars represent the mean ± SD of three independent experiments (***, P < 0.001). (C) H4 cells were either left untreated (lane 1) or transfected with a plasmid encoding HA-epitope-tagged EDEM1 (lanes 2 and 3). After 16 h cells were left untreated or treated with 250 µM IDN5706 for 8 h, and cellular extracts were subjected to SDS-PAGE followed by immunoblot with mouse antibody anti-HA-epitope. In A and C, Western blotting with antibody to ß-actin was used as loading control. The position of molecular mass markers is indicated on the left.
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