catalog no 13440s  (Cell Signaling Technology Inc)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Proteolysis-Targeting Chimeras Enhance T Cell Bispecific Antibody-Driven T Cell Activation and Effector Function through Increased MHC Class I Antigen Presentation in Cancer Cells.

doi: 10.4049/jimmunol.2000252

Figure Lengend Snippet: FIGURE 1. BRD4 degradation increases the abundance of endogenous MHC Ibound BRD4 peptides. (A) MAPPs workflow and BRD4 degrader tool compounds. (B) Protein levels of BRD4 on dBET1 or MZ1 degrader treatment for 8 h in SKM1 cells, as determined by Western blotting. Relative quantifica- tion to ACTIN is shown. (C) Relative peptide abundance of two BRD4 peptides binding to MHC I in SKM1 cells on treatment with either dBET1, MZ1, or DMSO for 8 h. Data are shown as mean of three peptide transitions ± SD. (D) Protein levels of BRD4 in SKM1 cells on dBET1 treatment over time, as determined by Western blotting. Relative quantification to ACTIN is shown. (E) Relative peptide abundance of two BRD4 peptides on MHC I in SKM1 cells on treatment with either 100 nM dBET1 or DMSO for 2 or 8 h. Data are shown as mean of two biological replicates ± SD. Each replicate reflects the mean of three peptide transitions.

Article Snippet: After blocking with Intercept (PBS) Blocking Buffer (catalog no. 927- 70001; LI-COR), the membrane was incubated in primary Ab (anti-BRD4, catalog no. 13440 [Cell Signaling]; anti-ACTIN, catalog no. 5441 [Sigma]; anti-MYC, catalog no. 950-25 [Invitrogen]; anti-WT1, ab89901 [Abcam]) at 4 C overnight, following the manufacturer’s instructions.

Techniques: Western Blot, Binding Assay, Quantitative Proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Proteolysis-Targeting Chimeras Enhance T Cell Bispecific Antibody-Driven T Cell Activation and Effector Function through Increased MHC Class I Antigen Presentation in Cancer Cells.

doi: 10.4049/jimmunol.2000252

Figure Lengend Snippet: FIGURE 2. BRD4 degrader-induced increase in MHC Ibound BRD4 peptide is dependent on proximity of E3 ligase and target protein. (A) Comparison of molecular structures of dBET1-(R), dBET1-NMe, JQ1, and thalidomide. (B) BRD4 protein expression levels in SKM1 cells on treatment with either dBET1, dBET1-(R), dBET1-NMe, JQ1, or thalidomide for 2 h. Relative quantification to ACTIN is shown. (C) Relative peptide abundance of two BRD4 peptides on MHC I in SKM1 cells that were treated with either dBET1, dBET1-(R), dBET1-NMe, JQ1, or thalidomide for 2 h. Data are shown as mean of three peptide transitions ± SD.

Article Snippet: After blocking with Intercept (PBS) Blocking Buffer (catalog no. 927- 70001; LI-COR), the membrane was incubated in primary Ab (anti-BRD4, catalog no. 13440 [Cell Signaling]; anti-ACTIN, catalog no. 5441 [Sigma]; anti-MYC, catalog no. 950-25 [Invitrogen]; anti-WT1, ab89901 [Abcam]) at 4 C overnight, following the manufacturer’s instructions.

Techniques: Comparison, Expressing, Quantitative Proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Proteolysis-Targeting Chimeras Enhance T Cell Bispecific Antibody-Driven T Cell Activation and Effector Function through Increased MHC Class I Antigen Presentation in Cancer Cells.

doi: 10.4049/jimmunol.2000252

Figure Lengend Snippet: FIGURE 3. BRD4 degradation increases the abundance of endogenous MHC Ibound BRD4 peptides in leukemia and melanoma cell lines. (A) Pro- tein expression levels on treatment with either dBET1 or DMSO for 4 h in CTV1 cells and A375 cells. (B) Relative peptide abundance of two MHC Ibound BRD4 peptides on treatment with either DMSO, 10 or 100 nM dBET1 for 2 h in SKM1 cells; DMSO, 1 or 100 nM dBET1 for 4 h in CTV1 cells; or DMSO, 100 or 1000 nM dBET1 for 4 h in A375 cells. Data are shown as mean of three peptide transitions ± SD, unless otherwise indicated.

Article Snippet: After blocking with Intercept (PBS) Blocking Buffer (catalog no. 927- 70001; LI-COR), the membrane was incubated in primary Ab (anti-BRD4, catalog no. 13440 [Cell Signaling]; anti-ACTIN, catalog no. 5441 [Sigma]; anti-MYC, catalog no. 950-25 [Invitrogen]; anti-WT1, ab89901 [Abcam]) at 4 C overnight, following the manufacturer’s instructions.

Techniques: Expressing