Pabg1 100

To detect the specific and efficiency of immunoprecipitation, 1/10 (10 µL) beads as Western blot (WB) samples were taken out from total IP beads with the bound target proteins. WB samples were firstly reduced and denatured by boiling in SDS buffer at 100 °C for 5 min, then loaded and isolated in SDS-PAGE gel. Next, the isolated proteins in gels were transfer into PVDF membrane using Trans-Blot^®SD semi-dry Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA) at 30 V for 25 min. For antibody staining, after 1 h blocking in blocking buffer (TBST buffer with 5% nonfat milk) at room temperature, the PVDF membrane with proteins was incubated overnight at 4 °C in the blocking buffer with 1:5000 diluted anti-GFP polyclonal antibody (PABG1-100, Chromotek). Then, washing the membrane using TBST for 3 × 5 min, and incubate the membrane in blocking buffer with the 1:5000 dilution of Goat Anti-Rabbit HRP conjugated secondary antibody (ab205718, Abcam, Cambridge, UK) at room temperature for 1 h. Wash the membrane three times again using TBST, 10 min each. For signal development, the membrane was covered with peroxide solution and the luminol/enhancer solution (SuperSignal West Femto, Thermo Fisher), then an image was acquired using a darkroom development instrument (DELIGHT-2010).

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)).