Figure S2.
TanGIBLE recognizes non-TMD model defective proteins. (A) Schematic of the CL1-fused luciferase model defective protein used in this study. The CL1 degron sequence was fused at the C-terminus of luciferase (Luc) and the N-terminus of Luc was fused with three repeated Flag-tags. This artificial proteasomal substrate was designated as Flag-Luc-CL1. (B) TanGIBLE co-precipitates more CL1-fused luciferase than BAG6 N200. After the expression of Flag-Luc-CL1 and a series of T7-tagged probes, HeLa cells were treated with 5 μM MG-132 for 4.5 h. Anti-T7 precipitates were blotted using anti-FLAG or anti-T7 antibodies. α-Tubulin was used as a loading control. (C) Quantification of the relative signal intensity of Flag-Luc-CL1 co-immunoprecipitated (IP) with probes. The value of the co-precipitated Flag signal with N200 fragment was defined as a standard (as 1.0), and the relative value with TanGIBLE is indicated. The graph represents the mean ± SD calculated from eight independent biological replicates (n = 8). The P value was calculated using the Student’s t test between N200 and TanGIBLE data. (D) Schematic representation of the mutations in the SS of prepro-insulin. A point mutation in the SS of R6C insulin was identified in pedigrees of type I diabetes (PNDM/MODY) with heterozygous mutations. SS are indicated in green. (E) An insulin R6C mutant accumulated as the prepro-form in proteasome-suppressed cells. HeLa cells expressing C-terminally Flag-tagged insulin WT or R6C were treated with (+) or without (−) 10 μM MG-132. At 4 h after MG-132 treatment, the cells were lysed and analyzed via immunoblotting using an anti-Flag antibody. (F) An insulin R6C mutant is unstable and degraded by the proteasome. HeLa cells expressing C-terminally Flag-tagged R6C insulin was chased with CHX in the presence or absence (DMSO) of 10 μM MG-132. The cells were lysed at the indicated time points and analyzed via immunoblotting using an anti-Flag-antibody. (G) TanGIBLE interacts with the ER-translocation-failed prepro-form of insulin mutants in proteasome-suppressed cells. HeLa cells expressing C-terminally T7-tagged insulin (WT or R6C) and Flag-tagged TanGIBLE were treated with 10 μM MG-132, as indicated. At 4 h after MG-132 treatment, the cells were lysed and immunoprecipitated with anti-Flag M2 agarose beads. Precipitates were blotted using the indicated antibodies. (H) Schematic representation of the human prion PrP and its SS mutants used in this study. Amino acid sequences of WT, N3a, and Prl (prolactin-type)-PrP SS are also provided. (I) Covalent modifications of N3a-PrP co-precipitated with TanGIBLE were weakened by the inhibition of ubiquitin-activating E1. MLN7243 was added to the cell cultures at 10 μM for 4 h before harvesting the cells (Takahashi et al., 2023). TanGIBLE immunoprecipitates from the cell lysates were probed with anti-T7 (PrP) antibody. The asterisk indicates the immunoglobulin signal. Source data are available for this figure: SourceData FS2.

TanGIBLE recognizes non-TMD model defective proteins. (A) Schematic of the CL1-fused luciferase model defective protein used in this study. The CL1 degron sequence was fused at the C-terminus of luciferase (Luc) and the N-terminus of Luc was fused with three repeated Flag-tags. This artificial proteasomal substrate was designated as Flag-Luc-CL1. (B) TanGIBLE co-precipitates more CL1-fused luciferase than BAG6 N200. After the expression of Flag-Luc-CL1 and a series of T7-tagged probes, HeLa cells were treated with 5 μM MG-132 for 4.5 h. Anti-T7 precipitates were blotted using anti-FLAG or anti-T7 antibodies. α-Tubulin was used as a loading control. (C) Quantification of the relative signal intensity of Flag-Luc-CL1 co-immunoprecipitated (IP) with probes. The value of the co-precipitated Flag signal with N200 fragment was defined as a standard (as 1.0), and the relative value with TanGIBLE is indicated. The graph represents the mean ± SD calculated from eight independent biological replicates (n = 8). The P value was calculated using the Student’s t test between N200 and TanGIBLE data. (D) Schematic representation of the mutations in the SS of prepro-insulin. A point mutation in the SS of R6C insulin was identified in pedigrees of type I diabetes (PNDM/MODY) with heterozygous mutations. SS are indicated in green. (E) An insulin R6C mutant accumulated as the prepro-form in proteasome-suppressed cells. HeLa cells expressing C-terminally Flag-tagged insulin WT or R6C were treated with (+) or without (−) 10 μM MG-132. At 4 h after MG-132 treatment, the cells were lysed and analyzed via immunoblotting using an anti-Flag antibody. (F) An insulin R6C mutant is unstable and degraded by the proteasome. HeLa cells expressing C-terminally Flag-tagged R6C insulin was chased with CHX in the presence or absence (DMSO) of 10 μM MG-132. The cells were lysed at the indicated time points and analyzed via immunoblotting using an anti-Flag-antibody. (G) TanGIBLE interacts with the ER-translocation-failed prepro-form of insulin mutants in proteasome-suppressed cells. HeLa cells expressing C-terminally T7-tagged insulin (WT or R6C) and Flag-tagged TanGIBLE were treated with 10 μM MG-132, as indicated. At 4 h after MG-132 treatment, the cells were lysed and immunoprecipitated with anti-Flag M2 agarose beads. Precipitates were blotted using the indicated antibodies. (H) Schematic representation of the human prion PrP and its SS mutants used in this study. Amino acid sequences of WT, N3a, and Prl (prolactin-type)-PrP SS are also provided. (I) Covalent modifications of N3a-PrP co-precipitated with TanGIBLE were weakened by the inhibition of ubiquitin-activating E1. MLN7243 was added to the cell cultures at 10 μM for 4 h before harvesting the cells (Takahashi et al., 2023). TanGIBLE immunoprecipitates from the cell lysates were probed with anti-T7 (PrP) antibody. The asterisk indicates the immunoglobulin signal. Source data are available for this figure: SourceData FS2.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal