Figure S3.
Related toFig. 4, newly synthesized ubiquitinated proteins are endogenous clients of TanGIBLE. (A) Hot lysis immunoprecipitation (IP) analysis showed that SDS denatured TanGIBLE does not co-precipitate ubiquitin moieties. Flag-tagged TanGIBLE, UBQLN4, and Rab8a T22N mutant (a positive control for covalently polyubiquitinated protein) were expressed in MG-132–treated HeLa cells. The cell lysates treated with 1% SDS denaturation at 90°C were diluted with buffer A that did not include SDS, and then the samples were affinity-purified with anti-Flag antibody gel beads. Bounded proteins after precipitations were subjected to western blot analysis with anti-polyubiquitin FK2 or anti-Flag antibodies. Asterisk indicates signals of immunogloblin. (B) CHX-chase experiment suggested that both TanGIBLE and its 10S mutant are stable proteins in HeLa cells. Tubulin was used as a loading control. (C) Related to Fig. 4 D, polyubiquitinated proteins associated with TanGIBLE are sensitive to CHX. HeLa cells expressing T7-tagged TanGIBLE, BAG6 FL, or N200 fragments were treated with 25 μg/ml CHX (or its solvent, EtOH) and 10 μM MG-132 as indicated for 4 h, then affinity-purified with an anti-T7 antibody from cell extracts. T7-precipitates (IP:T7) were blotted with anti-polyubiquitin FK2 antibody to detect co-precipitated endogenous polyubiquitinated proteins. Yellow asterisks indicate signals of FL BAG6 cross-reacted with FK2 antibody. (D and E) TanGIBLE expression did not perturb the successful synthesis of tail-anchored proteins in HeLa cells. The expression level of endogenous SEC61β, a translocon subunit, was not affected by TanGIBLE expression (D). Two tail-anchored proteins, SEC61β and RAMP1, were C-terminally fused with opsin (OPG)-tag, a glycosylation site (E). Successful assembly of these tail-anchored proteins in the ER stimulates glycosyl modifications. These opsin-tagged tail-anchored proteins were co-expressed with TanGIBLE in HeLa cells. Glycosylations of these tail anchored proteins were not affected by TanGIBLE co-expression (E), suggesting that TanGIBLE does not perturb tail-anchored protein biogenesis. (F) TanGIBLE immunoprecipitates and their input extracts from HCT116 and HeLa cells were probed with FK2 anti-polyubiqitin antibody. Source data are available for this figure: SourceData FS3.

Related to Fig. 4 , newly synthesized ubiquitinated proteins are endogenous clients of TanGIBLE. (A) Hot lysis immunoprecipitation (IP) analysis showed that SDS denatured TanGIBLE does not co-precipitate ubiquitin moieties. Flag-tagged TanGIBLE, UBQLN4, and Rab8a T22N mutant (a positive control for covalently polyubiquitinated protein) were expressed in MG-132–treated HeLa cells. The cell lysates treated with 1% SDS denaturation at 90°C were diluted with buffer A that did not include SDS, and then the samples were affinity-purified with anti-Flag antibody gel beads. Bounded proteins after precipitations were subjected to western blot analysis with anti-polyubiquitin FK2 or anti-Flag antibodies. Asterisk indicates signals of immunogloblin. (B) CHX-chase experiment suggested that both TanGIBLE and its 10S mutant are stable proteins in HeLa cells. Tubulin was used as a loading control. (C) Related to Fig. 4 D, polyubiquitinated proteins associated with TanGIBLE are sensitive to CHX. HeLa cells expressing T7-tagged TanGIBLE, BAG6 FL, or N200 fragments were treated with 25 μg/ml CHX (or its solvent, EtOH) and 10 μM MG-132 as indicated for 4 h, then affinity-purified with an anti-T7 antibody from cell extracts. T7-precipitates (IP:T7) were blotted with anti-polyubiquitin FK2 antibody to detect co-precipitated endogenous polyubiquitinated proteins. Yellow asterisks indicate signals of FL BAG6 cross-reacted with FK2 antibody. (D and E) TanGIBLE expression did not perturb the successful synthesis of tail-anchored proteins in HeLa cells. The expression level of endogenous SEC61β, a translocon subunit, was not affected by TanGIBLE expression (D). Two tail-anchored proteins, SEC61β and RAMP1, were C-terminally fused with opsin (OPG)-tag, a glycosylation site (E). Successful assembly of these tail-anchored proteins in the ER stimulates glycosyl modifications. These opsin-tagged tail-anchored proteins were co-expressed with TanGIBLE in HeLa cells. Glycosylations of these tail anchored proteins were not affected by TanGIBLE co-expression (E), suggesting that TanGIBLE does not perturb tail-anchored protein biogenesis. (F) TanGIBLE immunoprecipitates and their input extracts from HCT116 and HeLa cells were probed with FK2 anti-polyubiqitin antibody. Source data are available for this figure: SourceData FS3.

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