Rabbit anti calnexin

Cell media samples were concentrated 10 times using Vivaspin 500 columns (Sartorius Stedim Lab Ltd, Stonehouse, UK) for the APOB-100 detection. Cell lysates as well as concentrated and diluted cell media samples were mixed with Laemmli buffer containing 2-mercaptoethanol (ratio sample: Laemmli buffer = 4:1 v/v) and incubated for 5 min at 95 °C. Proteins were size-separated by SDS-page (6% acrylamide gel, 90 V, 3 h at 4 °C for APOB-100 and calnexin detection; 10% acrylamide gel, 130 V, 1 h at room temperature for albumin detection) and transferred onto a nitrocellulose membrane (0.2 A, 2.5 h at 4 °C). Nitrocellulose membranes were incubated overnight with primary antibodies, washed 3 times for 10 min with tris-buffered saline containing 0.2% tween (0.2% TBS-T buffer), incubated 1 h with HRP-conjugated secondary antibodies, washed 3 times for 10 min with 0.2% TBS-T buffer, incubated with Immobilon Western chemiluminescent HRP substrate (Millipore Corporation, Billerica, Massachusetts, USA). Bands were visualized using Chemidoc XRS System (Biorad, Hercules, California, USA). The following antibodies were used: mouse anti-APOB (Santa Cruz Biotechnology, Dallas, Texas, USA), rabbit anti-calnexin (Sigma-Aldrich, Saint Louis, Missouri, USA), mouse anti-albumin (Sigma-Aldrich, Saint Louis, Missouri, USA).

Tun, thapsigargin, ML385 and common reagents were from Sigma. GSK606414 was from APExBio and CCT020312 from Calbiochem. Mouse anti-actin and rabbit anti-calnexin antibodies were from Sigma. Mouse anti-eIF2α was from Cell Signaling. Rabbit anti-phospho-eIF2α (Ser51) was from MBL. Rabbit anti-PERK was from Abcam. Rabbit anti-ATF4 was a kind gift from David Ron (Univ. Cambridge). Goat anti-mouse IgG conjugated to 488 DyeLight, goat anti-rabbit IgG conjugated to 564 DyeLight, and goat anti-rabbit and anti-mouse IgG conjugated to HRP were from Jackson Labs. Normal goat serum was from Vector Laboratories (Burlingame, CA).

Protein extracts were prepared in RIPA buffer (10 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EGTA, 0.5% Triton and complete 1% protease and phosphatase inhibitor mixture, Roche Diagnostics). Primary neurons, brain and liver lysate samples (50 μg protein lysates) were separated using 8% polyacrylamide gel and then transferred to PVDF membranes. Membranes were incubated overnight at 4°C with the following primary antibodies in 1X PBST with 5% w/v nonfat dry: rabbit anti-MeCP2 (1:1000; Sigma-Aldrich, RRID:AB_262075), mouse anti-V5 (1:1000; ThermoFisher Scientific, RRID:AB_2556564), rabbit anti-pS6 235/236 (1:500, Cell Signaling, RRID:AB_331679), rabbit anti-S6 (1:500, Cell Signaling, RRID:AB_945319), rabbit anti-Calnexin (1:50000, Sigma-Aldrich, RRID:AB_476845), mouse anti-β-Actin (1:50000; Sigma-Aldrich, RRID:AB_262137) or the mice serum (1:200) extracted through retro-orbital bleeding followed by centrifugation (10 min, RT, 13000 rpm). Subsequently, membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000; Dako). The signal was then revealed with a chemiluminescence solution (ECL reagent, RPN2232; GE Healthcare) and detected with the ChemiDoc imaging system (Bio-Rad).