Anti flag coated magnetic beads

Cell culture medium and 1% penicillin/streptomycin were purchased from Welgene (Daegu, Korea). Fetal bovine serum (FBS) was purchase from Thermo Fisher Scientific (Waltham, MA, USA) and Atlas biologicals (Fort Collins, CO, USA). CHX, CIP and anti-FLAG-coated magnetic beads were purchased from Sigma (St. Louis, MO, USA). All vectors for cloning and transfection, Lipofectamine 3000, and blasticidin were purchased from Invitrogen (Carlsbad, CA, USA).

Relevant proteins were transiently expressed in HEK-293FT cells, followed by cell lysis in a buffer as described previously^{24 (link),25 (link)}. After cell lysis, anti-FLAG-coated magnetic beads (Sigma) or anti-His-tagged agarose beads (Qiagen, Hilden, Germany) were added, followed by further incubation at 4 °C for 2 h. The precipitated proteins were washed in the same lysis buffer and subjected to immunoblotting with anti-FLAG (1:2000; F3165, RRID: AB_259529) (Sigma), anti-XBP1 (1:2000; 619501, RRID: AB_319507) (Biolegend, San Diego, CA, USA) and anti-β-actin (1:4000; #8457, RRID: AB_10950489) (Cell Signaling, Danvers, MA, USA). Cells were also harvested at the indicated time points, and whole-cell-lysates were analyzed by immunoblotting with anti-XBP1 (Biolegend), anti-Fbw7 (1:2000; SC-293423), anti-p50 (NF-κB) (1:2000; SC-8414, RRID: AB_628015), anti-E2F-1 (1:2000; SC-251, RRID: AB_627476) (Santa Cruz Biotechnology, Dallas, TX, USA), anti-β-actin (Cell Signaling), and anti-Lamin B (1:2000; PA 50043) (AB Frontier, Seoul, Korea) antibodies.