Figure S4.
Related toFigs. 5 and 6, LC-MS/MS–based identification of defective proteins trapped by TanGIBLE. (A) Flag-TanGIBLE immunoprecipitates (IP) from MG-132–treated HeLa cell lysates were silver stained. Identical precipitates were anti-polyubiquitin immunoblotted in Fig. 5 A. The estimated protein concentration of each sample is noted at the bottom of the gel image. Note that Flag-TR-TUBE immunoprecipitated a much larger amount of protein, even though the protein expression level of Flag-TR-TUBE was below the limit of detection (Yoshida et al., 2015). (B) Immunoprecipitates shown in A were directly subjected to LC-MS/MS analysis. Volcano plot of TanGIBLE- or TR-TUBE–immunoprecipitates identified by deep MS analysis are shown. (C) TanGIBLE immunoprecipitates were treated with (+) or without (−) PGNase, a deglycosylation enzyme. Endogenous basigin in TanGIBLE precipitates (indicated by red arrowheads) was unaffected by deglycosylation treatment (compare lanes 5 and 6). Asterisk indicates signals of immunoglobulin. (D) TanGIBLE-associated basigin is sensitive to 4 h treatment with the protein synthesis inhibitor CHX. CHX was added to the cell culture 2 h prior to MG-132 addition. Defective basigins co-immunoprecipitated with TanGIBLE are indicated by red arrowheads. (E) Endogenous BST2 protein with no glycosyl modifications was co-immunoprecipitated with TanGIBLE. Note that successfully synthesized BST2 (indicated by a yellow line in the input lane) was not detected in the TanGIBLE precipitates (a yellow line in the IP lane). Note that TR-TUBE co-precipitated BST2 efficiently, supporting the observation that BST2 is a TUBE-preferred target protein (Fig. 5 F). Asterisk indicates a signal of immunoglobulin. (F) The MCM6 immunosignal that co-immunoprecipitated with TanGIBLE is specific since MCM6 siRNA canceled the corresponding signal in the TanGIBLE precipitates (compare lanes 1 and 2 in upper Flag IP panel). Tubulin is shown as a loading control for the input samples. (G) TanGIBLE-associated MCM6 is sensitive to protein synthesis inhibitor CHX. CHX was added to the cell culture 2 h prior to MG-132 addition. (H) Orphaned MCM5 subunit derived from disrupted MCM complex is a target of TanGIBLE. Although the total amount of MCM5 protein was greatly reduced by MCM2 siRNA (lower input panel, compare lanes 5 and 6), endogenous MCM5 co-precipitated with TanGIBLE was not affected by MCM2 depletion (upper IP panel). Note that TanGIBLE-associated MCM5 is highly sensitive to 4 h MG-132 treatment. Anti-MCM7 immunoblot signals shown in Fig. 6 D were obtained by re-probing with identical membrane used in this figure. Note that molecular weights of MCM7 and MCM5 were different. Source data are available for this figure: SourceData FS4.

Related to Figs. 5 and 6, LC-MS/MS–based identification of defective proteins trapped by TanGIBLE. (A) Flag-TanGIBLE immunoprecipitates (IP) from MG-132–treated HeLa cell lysates were silver stained. Identical precipitates were anti-polyubiquitin immunoblotted in Fig. 5 A. The estimated protein concentration of each sample is noted at the bottom of the gel image. Note that Flag-TR-TUBE immunoprecipitated a much larger amount of protein, even though the protein expression level of Flag-TR-TUBE was below the limit of detection (Yoshida et al., 2015). (B) Immunoprecipitates shown in A were directly subjected to LC-MS/MS analysis. Volcano plot of TanGIBLE- or TR-TUBE–immunoprecipitates identified by deep MS analysis are shown. (C) TanGIBLE immunoprecipitates were treated with (+) or without (−) PGNase, a deglycosylation enzyme. Endogenous basigin in TanGIBLE precipitates (indicated by red arrowheads) was unaffected by deglycosylation treatment (compare lanes 5 and 6). Asterisk indicates signals of immunoglobulin. (D) TanGIBLE-associated basigin is sensitive to 4 h treatment with the protein synthesis inhibitor CHX. CHX was added to the cell culture 2 h prior to MG-132 addition. Defective basigins co-immunoprecipitated with TanGIBLE are indicated by red arrowheads. (E) Endogenous BST2 protein with no glycosyl modifications was co-immunoprecipitated with TanGIBLE. Note that successfully synthesized BST2 (indicated by a yellow line in the input lane) was not detected in the TanGIBLE precipitates (a yellow line in the IP lane). Note that TR-TUBE co-precipitated BST2 efficiently, supporting the observation that BST2 is a TUBE-preferred target protein (Fig. 5 F). Asterisk indicates a signal of immunoglobulin. (F) The MCM6 immunosignal that co-immunoprecipitated with TanGIBLE is specific since MCM6 siRNA canceled the corresponding signal in the TanGIBLE precipitates (compare lanes 1 and 2 in upper Flag IP panel). Tubulin is shown as a loading control for the input samples. (G) TanGIBLE-associated MCM6 is sensitive to protein synthesis inhibitor CHX. CHX was added to the cell culture 2 h prior to MG-132 addition. (H) Orphaned MCM5 subunit derived from disrupted MCM complex is a target of TanGIBLE. Although the total amount of MCM5 protein was greatly reduced by MCM2 siRNA (lower input panel, compare lanes 5 and 6), endogenous MCM5 co-precipitated with TanGIBLE was not affected by MCM2 depletion (upper IP panel). Note that TanGIBLE-associated MCM5 is highly sensitive to 4 h MG-132 treatment. Anti-MCM7 immunoblot signals shown in Fig. 6 D were obtained by re-probing with identical membrane used in this figure. Note that molecular weights of MCM7 and MCM5 were different. Source data are available for this figure: SourceData FS4.

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