Fla 7000 phosphorimager

Wild‐type and ERp18 KO HEK293T cells stably overexpressing ATF6 were incubated in medium lacking methionine and cysteine for 30 min and pulse‐labeled for 30 min with 22 μCi/ml of EXPRESS35S Protein Labeling Mix (PerkinElmer). The cells were rinsed twice in PBS to remove the radiolabel and then incubated in complete medium to initiate the chase periods. Subsequently, cells were washed twice with PBS supplemented with 20 mM NEM and lysed in cell lysis buffer containing 20 mM NEM. Samples were subject to centrifugation at 16,000 × g at 4°C to obtain the post‐nuclear supernatant. ATF6α immunoisolation was carried out as described above using anti‐ATF6 (Abcam). Samples were separated under non‐reducing and reducing SDS–PAGE gels, fixed, dried, and exposed to a phosphorimager plate for 72 h. Radioactivity was detected using a Fujifilm FLA‐7000 Phosphorimager.

Southern blot on genomic DNA was performed as described in Thijssen et al.^{63 (link)}. For major satellite analysis 200 ng of genomic DNA were digested with HpyCH4IV (New England Biolabs) for 1h at 37 °C, while for minor satellites 500ng of gDNA were digested with HpaII (New England Biolabs) and 300 ng with MspI (New England Biolabs), both O/N at 37 °C. Digested samples were separated for 5 h on 1% agarose gel. Gels were then denaturated in a 1.5 M NaCl and 0.5 M NaOH solution for 20 min and neutralized with 0.5 M Tris-HCl pH 7.5 and 1.5 M NaCl for 40 min. Transfer was performed O/N on Hybond-N+ membranes (GE Healthcare) in SSC 20X. After ultraviolet crosslinking, membranes were pre-hybridized in SSC 6X, Denhardt 5X and 0.1% SDS for 1 h at 42 °C and hybridized with 32P-labelled probes for 2 h at 42 °C. After membrane washing, signals were detected using FLA 7000 phosphorimager (Fuji). Images were then analyzed with ImageJ (imagej.nih.gov/ij) to perform linescan for major satellites and intensity ratio HpaII/MspI for the lower six bands of each lane for minor satellites. Probe used: Major satellites 5′-CAC GTC CTA CAG TGG ACA TTT CTA AAT TTT CCA CCT TTT TCA GTT-3′ and minor satellites 5′-ACA TTC GTT GGA AAC GGG ATT TGT AGA ACA GTG TAT ATC AAT GAG TTA CAA TGA GAA ACA T-3′.

For pulse-label chase experiments, transfected Hek293T cells were starved for 60 min in methionine/cysteine free DMEM (Invitrogen) supplemented with 10% dialysed fetal calf serum, then metabolically labelled for 10 min with 55 μCi/ml ³⁵S-methionine/cysteine protein labeling mix (Perkin Elmer). Cells were rinsed with PBS, then chased in normal growth medium. At the end of the chase period cells were rinsed with PBS and solubilized in 1% Triton X-100 followed by immunoprecipitation of FLAG-tagged proteins as described below. Samples were analyzed by SDS-PAGE and labeled proteins were visualized by a FLA-7000 phosphorimager (Fuji).

Cells were starved for 30 min in cysteine/methionine-free DMEM and then radiolabeled in the same medium containing 22 μCi/ml EXPRESS³⁵S protein labeling mix (PerkinElmer Life Sciences). After 30 min of incubation at 37 °C, the radiolabel was removed, and cells were washed with PBS and incubated in complete DMEM for various lengths of time. Cells were washed with PBS before being lysed in buffer A. Cell debris was removed by centrifugation (10,000 × g for 10 min at 4 °C). The lysates were precleared by adding protein A-Sepharose (Generon) and incubated for 30 min at 4 °C. For immunoisolation, anti-PrxIV antibody was added to protein A-Sepharose. Samples were incubated for 16 h at 4 °C with end-over-end mixing. The Sepharose beads were pelleted by centrifugation (600 × g for 1 min) and washed three times with buffer A. Proteins were eluted with IEF sample buffer prior to loading onto a one-dimensional IEF gel. Gels were fixed, dried, and exposed to a phosphorimaging plate. Radioactivity was detected using a Fujifilm FLA-7000 PhosphorImager. Quantification of band intensities was carried out using ImageJ software.

Reactions were performed in TGMNKD buffer (10% (v/v) glycerol, 150 mM NaCl, 50 mM Tris HCl, 1 mM DTT, 5 mM MgCl₂, 50 mM KCl, pH 8.0) and initiated by the addition of 2 mM [γ³²P] ATP (3.7 GBq/mmol PerkinElmer). The reactions contained 5 μM GacS derivative and 20 μM RetS derivative (Fig. 1m, n). The final reaction volume was 100 µl; 10 µl aliquots were taken at the indicated timepoints and quenched in 20 µl of 2× SDS loading dye (7.5% (w/v) SDS, 90 mM EDTA, 37.5 mM Tris pH 6.8, 37.5% glycerol, and 3% β-mercaptoethanol). Samples were stored on ice and then analysed using SDS-PAGE (10% (w/v) polyacrylamide). Gels were exposed to phosphorscreens (Fuji) for 1 h and then analysed using a Fujifilm FLA-7000 phosphorimager. The uncropped phosphorimages used to produce Fig. 1 and Supplementary Fig. 1 are shown in Supplementary Figs. 5 and 6.