Purification of Organelles from Activated B Cells

The procedure was performed as described previously^{33 (link),54 (link)}. Briefly, activated B cells from Tent5c-GFP knock-in mice were lysed in MTE buffer (270 mM D-mannitol, 10 mM Tris pH 7.4, 0.1 mM EDTA, 1 mM PMSF) by homogenization monitored by microscopy. Extracts were cleared by sequential centrifugation at 700 and 15,000 × g and subsequently were loaded on the top of the discontinuous sucrose gradient (1.3 M, 1.5 M, and 2 M prepared in 10 mM Tris pH 7.6, 0.1 mM EDTA) and ultracentrifuged at 152,000 × g for 70 min in an SW41 rotor (Beckman). The top layer was collected as cytosol fraction. ER fraction was collected as band at the interphase of 1.3 M sucrose layer, diluted with additional MTE buffer and ultracentrifuged at 126,000 × g for 45 min in MLA130 rotor (Beckman), and pellet was collected as purified ER, resuspended in PBS supplemented with proteases inhibitors and subsequently analyzed using western blot and antibodies against TRAPα (Clone EPR5603), SSR3 (Abcam, ab190936), HDLBP (Bethyl, A303-971A), and PERK (Clone C33E10, GRP94 (Clone H-212), CHOP (Clone D46F1), and GFP (ChromoTek, PABG1-100)).