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A
In vitro ubiquitylation of IRE1α by MITOL. In vitro ubiquitylation assay was performed as described in methods. Immunoprecipitated IRE1α‐FLAG from HEK293 cells was incubated with or without indicated materials, followed by immunoblotting with indicated antibodies.
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B
MITOL did not affect the protein turnover of IRE1α. MEFs were incubated with 10 μg/ml cycloheximide (CHX) for indicated periods, followed by immunoblotting with indicated antibodies. Error bars represent SD (n = 3). n.s.: not significant (Student's t‐test).
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C, D
ER stress attenuated IRE1α ubiquitylation by MITOL and enhanced the interaction between IRE1α and BIM. MITOL‐KO MEFs were transfected with indicated vectors 24 h prior to Tu treatment for indicated periods. Cell lysates were subjected to immunoprecipitated with indicated antibodies, followed by immunoblotting with indicated antibodies.
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E
The levels of cleaved caspase‐3 of MEFs after Tu treatment. MEFs were transfected with empty vector or MITOL‐coding vector 24 h prior to Tu treatment. Tu was treated for indicated periods, followed by immunoblotting with indicated antibodies.
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F
Autoubiquitylation activity of MITOL was not changed under ER stress. HEK293 cells were transfected with indicated vectors 24 h prior to Tu treatment for 12 h, followed by IP‐IB analysis with indicated antibodies.
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G
MITOL preferentially ubiquitylated IRE1α monomer. HEK293 cells were transfected with indicated vectors, followed by IP‐IB analysis with indicated antibodies. K121Y (KY) or D123P (DP) mutant of IRE1α inhibits its self‐association.
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H–J
Inhibition of IRE1α RNase enhanced MITOL‐dependent IRE1α ubiquitylation. HEK293 cells were transfected with indicated vectors 24 h prior to incubation with either 10 μM 4μ8C, 1 μM KIRA6, or 2 μM APY‐29 for 3 h, followed by IP‐IB analysis with indicated antibodies.
