Calreticulin

Cell lysates were prepared with an isotonic buffer (10 mM HEPES, 5 mM MgCl₂, 1 mM EGTA, 142 mM KCl with NP-40), in the presence of complete, EDTA-free, protease inhibitor cocktail (Roche). Samples were clarified, denatured with SDS buffer, and boiled. Proteins were separated by SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane (Biorad). Blots were probed with antibodies against caspase-11 (Sigma), caspase-1 (Genentech), phospho-cofilin, cofilin, Rac/Cdc42, and phospho-Rac (Cell Signaling), actin (Abcam), calreticulin (Stressgen). Detection was achieved using appropriate secondary antibodies conjugated with horseradish peroxidase (HRP), as previously described12 (link)14 (link)22 (link)79 (link).

Rabbit polyclonal antibodies were used to detect ERdj3, EDEM1, and ERp57 (Proteintech, Chicago, IL); others were to detect AAT (DAKO, Carpentaria, CA), GAPDH (Santa Cruz, Dallas, TX), Lamp1, P62, LC3B (all from Cell Signaling, Danvers, MA), and calnexin and calreticulin (both from Stressgen biotechnologies, San Diego, CA). Bafilomycin A1, MG132, Brefeldin A, and mouse monoclonal anti‐β‐actin were purchased from Sigma (St. Louis, MO), and 2C1 against human AAT polymers was purchased from Hycult biotech (Netherlands). Mouse monoclonal antibodies against BiP and AAT were purchased from BD bioscience (San Jose, CA) and R&D systems (Minneapolis, MN), respectively. Alexa Fluor 488 goat anti‐mouse IgG and Alexa Fluor 594 goat anti‐rabbit IgG were purchased from Invitrogen. Primers, Dynabeads protein A and G, Superscript VILO cDNA synthesis kit, and ERdj3 siRNA were purchased from Life Technologies, and HP DNA transfection reagent and TaqMan Universal PCR Master Mix were purchased from Roche Applied Science. Disuccinimidyl suberate (DSS) cross‐linker was purchased from Thermo Fisher scientific (Waltham, MA).