Supplementary Figure 11: R-BiP is induced by prolonged proteasomal inhibition and colocalizes with ubiquitin conjugates in autophagic vacuoles. | Nature Cell Biology

Supplementary Figure 11: R-BiP is induced by prolonged proteasomal inhibition and colocalizes with ubiquitin conjugates in autophagic vacuoles.

From: Amino-terminal arginylation targets endoplasmic reticulum chaperone BiP for autophagy through p62 binding

Supplementary Figure 11

(a) R-BiP is induced by prolonged proteasomal inhibition. HeLa cells were treated with a proteasomal inhibitor or other stressors, followed by immunoblotting analysis of R-BiP and ubiquitin conjugates (as visualized using FK1 antibody). A23187, calcium ionophore; CCCP, carbonyl cyanide m-chlorophenylhydrazone (Protonophore (H+ ionophore) and uncoupler of oxidative phosphorylation in mitochondria). (b) R-BiP colocalizes with ubiquitin conjugates in autophagic vacuoles. Poly(dA:dT)-treated HeLa cells were subjected to immunostaining analysis of R-BiP and p62 with ubiquitin conjugates as visualized by FK2 antibody. This assay reveals that ubiquitin-positive puncta are invariably positive for both R-BiP and p62. Scale bar, 10 μm. (c) ATE1-knockdown inhibits Nt-arginylation of R-BiP as well as autophagic induction in HeLa cells treated with both 10 μM MG132 and 200 nM thapsigargin. (d) BiP-knockdown inhibits Nt-arginylation of R-BiP as well as autophagic induction in HeLa cells treated with both 10 μM MG132 and 200 nM thapsigargin. (e) R-BiP induced by proteasomal inhibition and ER stress is mainly retrieved from the cytosolic fraction.

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