Irdye fluorescently labelled secondary antibodies

After separation by SDS–PAGE or native-PAGE^{19 (link)} proteins were transferred onto PVDF membranes. The membranes were blocked with 5% (w/v) dried skimmed milk in TBS (25 mM Tris–HCl pH 7.5, 150 mM NaCl) and incubated with primary antibodies diluted in 3% (w/v) BSA in TBS supplemented with 0.1% Tween-20 (TBST). Primary antibodies and antisera against hamster BiP [chicken anti-BiP^{68 (link)}], eIF2α [mouse anti-eIF2α^{69 (link)}] and FICD [chicken anti-FICD^{3 (link)}] were used at a dilutions of 1/1000, 1/5000 and 1/1000 (v/v), respectively. Following the primary antibody incubation, the PVDF membrane was washed with TBST and then incubated with IRDye fluorescently labelled secondary antibodies (LI-COR) at a dilution of 1/2000 (v/v) in a solution of 3% (w/v) dried skimmed milk in TBS. The membranes were scanned with an Odyssey near-infra-red imager (LI-COR). Where applicable, IB band quantification was carried out with Image Studio Lite software (LI-COR).

After separation by SDS-PAGE, the proteins were transferred onto PVDF membranes. Membranes were blocked with 5% (w/v) dried skimmed milk in TBS (25 mM Tris-HCl pH 7.5, 150 mM NaCl) and incubated with primary antibodies followed by IRDye fluorescently labelled secondary antibodies (LI-COR). Membranes were scanned with an Odyssey near-infrared imager (LI-COR). Primary antibodies against hamster BiP (chicken anti-BiP, 1:2000;^{48 (link)}), actin (mouse anti-actin, 1:2000; Abcam, cat. # AB3280), MANF (chicken anti-MANF, 1:1000; see below), FLAG-M1 (mouse anti-FLAG-M1, 1:1000; Sigma, cat. # F3040), and A1AT (mouse anti-A1AT monoclonal, 1:5000; Abcam, cat. # AB9399) were used.

After separation by SDS-PAGE on standard polyacrylamide Tris-glycine gels, the proteins were transferred onto PVDF membranes (pore size 0.45 μm, Sigma). The membranes were blocked with 5% (w/v) dried skimmed milk in TBS (25 mM Tris-HCl pH 7.5, 150 mM NaCl) and incubated with primary antibodies followed by IRDye fluorescently labelled secondary antibodies (LI-COR). The membranes were scanned with an Odyssey near-infrared imager (LI-COR). Primary antibodies and antisera against human IRE1α LD (Shemorry et al, 2019 (link)), mouse IRE1α serum (NY200) (Bertolotti et al, 2000 (link)), hamster BiP (chicken anti-BiP (Avezov et al, 2013 (link))), eIF2α (mouse anti-eIF2α (Scorsone et al, 1987 (link))) and monoclonal anti-FLAG-M1 (Sigma) were used.
Coomassie-staining was carried out with Instant Blue (Expedeon). Signal quantitation from SDS-PAGE gels or from immunoblots was carried out using the ImageJ software (NIH). For quantitative immunoblotting (Fig. EV5B), a precast gel NuPAGE™ 4 to 12%, Bis-Tris, 1.0–1.5 mm, Mini Protein Gel (Thermo Fisher) was used.