Figure 4. Comparison of S-reactive electrophiles demonstrating that compound 147 selectively modifies ER-localized proteins.

(A) Proportion of ER-localized proteins covalently labeled by 147 or fragment electrophiles reported in (Backus et al., 2016). The ER-localized proportion for each electrophile indicated in red. The numbers in parentheses indicate the number of ER-localized proteins in a given electrophile’s target list on the left and the total number of proteins in the target list on the right. The electrophiles from (Backus et al., 2016) are denoted with a B- followed by the compound numbers used in that work. (B) Bar graph showing activation of the ERSE.FLuc reporter in HEK293T cells treated with 147 (10 µM), thapsigargin (Tg; 0.5 µM) or the indicated dose of acetaminophen for 18 hr. Error bars show SEM for 3 independent experiments. (C) Immunoblot of PDI4 in 147–20 affinity purified proteins from HEK293T cells treated with 147 (10 µM), or 147–20 (10 µM), or the combination of 147–20 (10 µM) and 147 (50 µM), or the combination of 147–20 (10 µM) and 147–4 (50 µM) for 18 hr. The relative recovery of PDIA4 under these different conditions is indicated below the immunoblot.

Figure 4—figure supplement 1. Compound 147 selectively modifies ER-localized proteins.

Graph showing activation of the ERSE.FLuc reporter in HEK293T cells treated with 147 (10 µM), thapsigargin (Tg; 0.5 µM) or the indicated dose of eugenol for 18 hr. Error bars show SEM for 3 independent experiments.

Figure 4—figure supplement 2. Compound 147 preferentially partitions to the ER.

(A) Western blot for ERdj3 (an ER-resident protein) and HSC70 (a cytosolic protein) in the ER and cytosolic fractions from a digitonin extraction of HEK293T cells after 15 min of treatment with 147 (10 μM). (B) Enrichment of 147 as determined by mass spectrometry in the cytosolic fraction versus the ER fraction of HEK293T cells after 15 min of treatment with 147 (10 µM). Error bars show SEM for 3 independent experiments.