Mg132 mg

Neuro2a cells were maintained in Dulbecco’s Modified Eagle’s Minimum Essential Medium containing 5% fetal bovine serum. Transfection of the indicated constructs was performed using the PEI-MAX reagent (Polysciences) as previously described^{5 (link),17 (link)}. For the establishment of CRELD2-deficient cells, Neuro2a cells transfected with the indicated constructs; the gRNA, hCas9 and donor genes, were cultured with hygromycin, and the resultant cells were used in this study^{17 (link)}. During these selections, the parental wild-type (WT) Neuro2a cells were maintained with the normal culture medium and were used as control cells for the following experiments. A CRELD2-deficient single clone was obtained after the seeding and growth of a cell in 96-well plate. In each experiment, parental and each deficient cell were seeded into 96- or 12-well plates or 3.5-cm dishes with non–hygromycin containing culture medium. After that, the cells were treated with or without thapsigargin (Tg, 0.1 μM), tunicamycin (Tm, 1 μg/ml), breferdin A (BFA, 2.5 μg/ml), cycloheximide (CHX, 10 μg/ml) (Sigma-Aldrich), MG132 (MG, 10 μM) (Peptide Institute) or serum-deprived medium (serum free, SF) for the indicated time period.

Thapsigargin (Tg), tunicamycin (Tm), brefeldin A (BFA), cycloheximide (CHX), and kifunensine (Kif) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Monensin (Mone) and nigericin (Nigr) were purchased from Abcam (Cambridge, UK). MG132 (MG) and concanamycin A (CMA) were obtained from Peptide Institute (Osaka, Japan) and Wako (Osaka, Japan), respectively.