"""Comparison of the differential susceptibility of unmodified and AMPylated BiP to cleavage by SubA in vitro. (A) Schema of the experimental design. ATP hydrolysis-deficient BiP T229A protein was AMPylated in presence of radioactive α- 32 P-ATP with catalytically active GST-FICD E234G coupled to GSH-Sepharose beads. The enzyme-containing beads were removed by centrifugation and excess of non-radioactive ATP was added to the supernatant to competitively displace non-covalently bound α- 32 P-ATP from BiP T229A . Unbound nucleotides were then removed by passing the sample through a desalting column. Trace amounts (1.5 µg) of the recovered 32 P-labeled AMPylated BiP T229A protein were added to excess of unmodified BiP T229A protein (60 µg; a mass ratio of 40:1) and the combined sample was supplemented with ATP and treated for 30 min with increasing concentrations of the SubA protease (0.08 to 120 ng/µl) before denaturing SDS-PAGE, Coomassie staining and autoradiography. ( B ) Autoradiograph and Coomassie stain (CBB) of an SDS-PAGE gel of BiP from samples described above. In the bottom panel, the Coomassie stain and radioactive signals of the full-length BiP and the substrate binding domain (SBD) were quantified and normalized to the values in lane 1, which were set arbitrarily to 1 for full-length BiP and to 0 for the SBD (graph). The Coomassie stain signal reports of the fate of unmodified BiP T229A , whereas the radioactive signals report exclusively on the modified BiP T229A in the combined sample. NBD denotes the nucleotide binding domain. ( C ) A shorter exposure (1 hr versus 8 hr) of the autoradiograph shown in """"B"""", above. It reveals the substantial conservation of the radioactive signal emanating from intact BiP across the time course (the green plot in """"B"""" above), which is obscured by changes in the band width introduced by the progressive digestion of the unlabeled (and unmodified) intact BiP in the sample. DOI: http://dx.doi.org/10.7554/eLife.12621.007"""
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