American Society of Hematology

Figure 2.

$Figure 2. CDK9 binds to protein components of mTOR complexes. (A) U937 cells were lysed, and mLST8 was immunoprecipitated with magnetic beads preconjugated with an anti-mLST8 antibody. Rabbit IgG preconjugated beads were used as negative controls for nonspecific binding. Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. mTOR was used as a positive control for binding. (B) HA-mLST8 was coexpressed with various FLAG-CDK9 acetylation mutants in 293T cells. Cells were lysed in (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate) (CHAPS) buffer, and FLAG-CDK9 was immunoprecipitated with Sepharose beads preconjugated with an anti-FLAG-M2 antibody. Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (C) HA-mLST8 or HA-RAPTOR was coexpressed with FLAG-CDK9 in 293T cells. Cells were lysed in CHAPS buffer, and FLAG-CDK9 was immunoprecipitated with Sepharose beads preconjugated with an anti-FLAG-M2 antibody. Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (D) MYC-RICTOR or MYC-mSIN1 was coexpressed with FLAG-CDK9 in 293T cells. Cells were lysed in CHAPS buffer, and FLAG-CDK9 was immunoprecipitated with Sepharose beads preconjugated with an anti-FLAG-M2 antibody. Empty vector coexpressed with MYC-RICTOR or MYC-SIN1 was used a negative control for nonspecific binding. Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (E) YFP-mTOR was coexpressed with FLAG-CDK9 in 293T cells. Cells were lysed in CHAPS buffer, and FLAG CDK9 was immunoprecipitated with Sepharose beads preconjugated with an anti–FLAG-M2 antibody. Empty vector coexpressed with YFP-mTOR was used as a negative control for nonspecific binding. Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (F) U937 cells were fractionated into cytoplasmic and nuclear lysates. Proteins were resolved by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were then immunoblotted with antibodies for tubulin (cytoplasmic marker) and lamin A/C (nuclear marker) to verify cellular fractionation. IP with magnetic beads preconjugated to an anti-CDK9 antibody was performed on cytoplasmic and nuclear cell lysates from U937 cells. Rabbit IgG preconjugated beads were used as negative controls for nonspecific binding. Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.$

CDK9 binds to protein components of mTOR complexes. (A) U937 cells were lysed, and mLST8 was immunoprecipitated with magnetic beads preconjugated with an anti-mLST8 antibody. Rabbit IgG preconjugated beads were used as negative controls for nonspecific binding. Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. mTOR was used as a positive control for binding. (B) HA-mLST8 was coexpressed with various FLAG-CDK9 acetylation mutants in 293T cells. Cells were lysed in (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate) (CHAPS) buffer, and FLAG-CDK9 was immunoprecipitated with Sepharose beads preconjugated with an anti-FLAG-M2 antibody. Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (C) HA-mLST8 or HA-RAPTOR was coexpressed with FLAG-CDK9 in 293T cells. Cells were lysed in CHAPS buffer, and FLAG-CDK9 was immunoprecipitated with Sepharose beads preconjugated with an anti-FLAG-M2 antibody. Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (D) MYC-RICTOR or MYC-mSIN1 was coexpressed with FLAG-CDK9 in 293T cells. Cells were lysed in CHAPS buffer, and FLAG-CDK9 was immunoprecipitated with Sepharose beads preconjugated with an anti-FLAG-M2 antibody. Empty vector coexpressed with MYC-RICTOR or MYC-SIN1 was used a negative control for nonspecific binding. Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (E) YFP-mTOR was coexpressed with FLAG-CDK9 in 293T cells. Cells were lysed in CHAPS buffer, and FLAG CDK9 was immunoprecipitated with Sepharose beads preconjugated with an anti–FLAG-M2 antibody. Empty vector coexpressed with YFP-mTOR was used as a negative control for nonspecific binding. Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (F) U937 cells were fractionated into cytoplasmic and nuclear lysates. Proteins were resolved by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were then immunoblotted with antibodies for tubulin (cytoplasmic marker) and lamin A/C (nuclear marker) to verify cellular fractionation. IP with magnetic beads preconjugated to an anti-CDK9 antibody was performed on cytoplasmic and nuclear cell lysates from U937 cells. Rabbit IgG preconjugated beads were used as negative controls for nonspecific binding. Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.

This Feature Is Available To Subscribers Only