Journal: eLife

Article Title: Pharmacologic ATF6 activating compounds are metabolically activated to selectively modify endoplasmic reticulum proteins

doi: 10.7554/eLife.37168

Figure Lengend Snippet: Protein targets that are covalently modified by 147 . 10.7554/eLife.37168.009 Table 1—source data 1. Excel spreadsheet showing the competition ratio data for all 90 proteins that were identified as targets of 147 in every replicate and every cell type. 10.7554/eLife.37168.010 Table 1—source data 2. Excel spreadsheet showing all the proteins identified among the affinity-purified targets of 147 .

Article Snippet: Proteins were transferred from gel slabs to nitrocellulose and blotted using mouse anti-FLAG M2 antibody (Sigma) or rabbit anti-PDIA4 antibody (Protein Tech) and visualized on the Odyssey Infrared Imaging System (Li-Cor Biosciences).

Techniques: Modification, Affinity Purification

Journal: eLife

Article Title: Pharmacologic ATF6 activating compounds are metabolically activated to selectively modify endoplasmic reticulum proteins

doi: 10.7554/eLife.37168

Figure Lengend Snippet: ( A ) Proportion of ER-localized proteins covalently labeled by 147 or fragment electrophiles reported in . 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 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.

Article Snippet: Proteins were transferred from gel slabs to nitrocellulose and blotted using mouse anti-FLAG M2 antibody (Sigma) or rabbit anti-PDIA4 antibody (Protein Tech) and visualized on the Odyssey Infrared Imaging System (Li-Cor Biosciences).

Techniques: Labeling, Activation Assay, Western Blot, Affinity Purification

Journal: eLife

Article Title: Pharmacologic ATF6 activating compounds are metabolically activated to selectively modify endoplasmic reticulum proteins

doi: 10.7554/eLife.37168

Figure Lengend Snippet: ( A ) Mechanistic model of 147 -dependent ATF6 activation, as described in the main text. The red component of the figure shows how pharmacologic modulation of ATF6 by 147 or CP7 influences ATF6 activity. ( B ) Immunoblot showing lysates prepared from HeLa cells stably expressing FLAG-tagged ATF6 treated for 6 hr with 147 (10 µM) or tunicamycin (Tm; 1 mg/mL). Lysates were separated by non-reducing or reducing SDS-PAGE prior to immunoblotting. The bands representing oxidized and reduced ATF6 are indicated. Note the increased ATF6 migration observed in reducing gels for Tm-treated cells, reflecting the inhibited N-linked glycosylation of ATF6 in these samples. ( C ) Graph showing relative activation of the ERSE.Fluc ATF6 reporter in HEK293T cells co-treated for 18 hr with 147 (10 µM) and increasing concentrations of CP7 , as indicated. Error bars show SEM for three technical replicates. ( D ) Bar graph showing BiP expression in HEK293T cells stably expressing non-silencing, PDIA1 , PDIA3, PDIA4 , PDIA5 , or PDIA6 shRNA, as indicated. BiP expression in the PDI-depleted cells is shown relative to cells expressing non-silencing shRNA. Error bars show SEM for 3 independent experiments. *indicates p<0.05. ( E ) Bar graph showing BiP expression in HEK293T cells expressing non-silencing, PDIA1 , PDIA3, PDIA4 , PDIA5 , or PDIA6 shRNA treated for 6 hr with or without 147 (10 µM). BiP expression levels for samples treated with 147 are normalized to the corresponding vehicle-treated controls. Un-normalized data are shown in . Error bars show SEM for 3 independent experiments. *indicates p<0.05. ( F ) Bar graph showing BiP expression in HEK293T cells expressing non-silencing, PDIA1 , PDIA3, PDIA4 , PDIA5 , or PDIA6 shRNA treated for 6 hr with or without thapsigargin (Tg; 0.5 µM). BiP expression levels for samples treated with Tg are normalized to the corresponding vehicle-treated controls. Un-normalized data are shown in Error bars show SEM for 3 independent experiments.

Article Snippet: Proteins were transferred from gel slabs to nitrocellulose and blotted using mouse anti-FLAG M2 antibody (Sigma) or rabbit anti-PDIA4 antibody (Protein Tech) and visualized on the Odyssey Infrared Imaging System (Li-Cor Biosciences).

Techniques: Activation Assay, Activity Assay, Western Blot, Stable Transfection, Expressing, SDS Page, Migration, Glycoproteomics, shRNA

Journal: eLife

Article Title: Pharmacologic ATF6 activating compounds are metabolically activated to selectively modify endoplasmic reticulum proteins

doi: 10.7554/eLife.37168

Figure Lengend Snippet: ( A ) Bar graph showing expression of PDIA1 , PDIA3 , PDIA4 , PDIA5 , or PDIA6 in HEK293T cells depleted for the same PDI. The dashed line reflects expression of each PDI in control cell lines expressing non-silencing shRNA. Error bars show ± 95% confidence interval. ( B ), ( C ) Un-normalized data for and , respectively. Error bars show SEM for 3 independent experiments.

Article Snippet: Proteins were transferred from gel slabs to nitrocellulose and blotted using mouse anti-FLAG M2 antibody (Sigma) or rabbit anti-PDIA4 antibody (Protein Tech) and visualized on the Odyssey Infrared Imaging System (Li-Cor Biosciences).

Techniques: Expressing, Control, shRNA