Figure 6.
Endogenous orphaned subunits derived from MCM complex are targets of TanGIBLE. (A, a–c) Heat maps of the subunits of CCT/TRiC complex (a), RNA polymerase II complex (b), and MCM complex (c), which were identified by MS analysis of TanGIBLE immunoprecipitates (IP). All subunits in the precipitates were augmented in the presence of MG-132 but were decreased with 10S mutations of the TanGIBLE probe. Panel colors show the relative fold increase compared with the base line (defined as 1.0). Schematic of MCM hexamer complex model (d). Reported hydrophobic interactions are indicated as red dots. (B) HeLa cells expressing 3xFlag-tagged TanGIBLE were lysed and immunoprecipitated with anti-Flag M2 antibody and the eluted endogenous proteins were simultaneously immunoblotted with anti-basigin and anti-MCM6 antibodies. Defective basigin (indicated by a black arrowhead) and MCM6 (indicated by a red arrowhead) were detected in WT TanGIBLE with MG-132 treatment. Note that 10S TanGIBLE did not co-precipitate defective basigin and endogenous MCM6. (C) Orphaned MCM6 subunit derived from disrupted complexes is a target of TanGIBLE. The total amount of MCM6 protein was greatly reduced by MCM2 siRNA (compare lanes 2 and 3 in input panel), whereas endogenous MCM6 co-precipitated with TanGIBLE is augmented by MCM2 depletion (compare lanes 2 and 3 in Flag IP panel). Note that MG-132 treatment did not restore the total amount of MCM6 protein significantly because MG-132–treated time (4 h) was too short to restore the total amount of MCM6 compared with the long period of MCM2 knockdown (72 h). Tubulin is shown as a loading control for input samples. (D) Augmentation of the endogenous MCM7 subunit level in a TanGIBLE immunoprecipitate after MCM2 siRNA treatment (compare lanes 5 and 6 in Flag IP panel), even though the total amount of MCM7 protein is greatly reduced by MCM2 siRNA (compare lanes 5 and 6 in input panel). Note that TanGIBLE-associated MCM7 is MG-132 sensitive (compare lanes 4 and 6 in Flag IP panel), while total MCM7 (shown in input panel) did not respond to short-term MG-132 treatment (4 h). Source data are available for this figure: SourceData F6.

Endogenous orphaned subunits derived from MCM complex are targets of TanGIBLE. (A, a–c) Heat maps of the subunits of CCT/TRiC complex (a), RNA polymerase II complex (b), and MCM complex (c), which were identified by MS analysis of TanGIBLE immunoprecipitates (IP). All subunits in the precipitates were augmented in the presence of MG-132 but were decreased with 10S mutations of the TanGIBLE probe. Panel colors show the relative fold increase compared with the base line (defined as 1.0). Schematic of MCM hexamer complex model (d). Reported hydrophobic interactions are indicated as red dots. (B) HeLa cells expressing 3xFlag-tagged TanGIBLE were lysed and immunoprecipitated with anti-Flag M2 antibody and the eluted endogenous proteins were simultaneously immunoblotted with anti-basigin and anti-MCM6 antibodies. Defective basigin (indicated by a black arrowhead) and MCM6 (indicated by a red arrowhead) were detected in WT TanGIBLE with MG-132 treatment. Note that 10S TanGIBLE did not co-precipitate defective basigin and endogenous MCM6. (C) Orphaned MCM6 subunit derived from disrupted complexes is a target of TanGIBLE. The total amount of MCM6 protein was greatly reduced by MCM2 siRNA (compare lanes 2 and 3 in input panel), whereas endogenous MCM6 co-precipitated with TanGIBLE is augmented by MCM2 depletion (compare lanes 2 and 3 in Flag IP panel). Note that MG-132 treatment did not restore the total amount of MCM6 protein significantly because MG-132–treated time (4 h) was too short to restore the total amount of MCM6 compared with the long period of MCM2 knockdown (72 h). Tubulin is shown as a loading control for input samples. (D) Augmentation of the endogenous MCM7 subunit level in a TanGIBLE immunoprecipitate after MCM2 siRNA treatment (compare lanes 5 and 6 in Flag IP panel), even though the total amount of MCM7 protein is greatly reduced by MCM2 siRNA (compare lanes 5 and 6 in input panel). Note that TanGIBLE-associated MCM7 is MG-132 sensitive (compare lanes 4 and 6 in Flag IP panel), while total MCM7 (shown in input panel) did not respond to short-term MG-132 treatment (4 h). Source data are available for this figure: SourceData F6.

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