brd9  (Cell Signaling Technology Inc)

Journal: Molecular Therapy

Article Title: A BPTF-specific PROTAC degrader enhances NK cell-based cancer immunotherapy

doi: 10.1016/j.ymthe.2025.02.013

Figure Lengend Snippet: 8d selectively degrades BPTF in a proteasome-dependent manner (A) Immunoblotting of BPTF, HPSE, CECR2 and BRD9 in the total cell lysates. The lysates were isolated from Huh7 cells treated with BPTF PROTAC degraders ( 8a-d ) at 10 μM for 24 h, respectively. β-Actin was used as the loading control. The displayed image is representative of three biological replicates. (B) Immunoblotting of BPTF and HPSE in the total cell lysates. The lysates were isolated from Huh7 cells treated with 8d at the indicated doses (0, 0.5, 1, 5, 10, and 25 μM) for 24 h β-actin was used as the loading control. The displayed image is representative of three biological replicates. (C) Degradation curves of BPTF in 8d -treated Huh7 cells. The expression level of BPTF was normalized to the level of β-actin. n = 3. (D) Immunoblotting of BPTF and HPSE in the total cell lysates. The lysates were isolated from Huh7 cells treated with 8d at 10 μM for the indicated times (0, 6, 12, 24, and 48 h). β-Actin was used as the loading control. The displayed image is representative of three biological replicates. (E) Volcanic plot illustrating the altered proteins between control and 8d -treated Huh7 cells. The significantly downregulated and upregulated proteins are represented by blue and red dots, respectively. (Two-sided paired t test, p < 0.05, FC > 1.5). (F) Venn diagram showing the similarities and differences between 8d -treated and BPTF KD Huh7 cell groups. (G) Immunoblotting of BPTF in the total cell lysates. The lysates were isolated from Huh7 cells that were pretreated with 8d (10 μM) for 24 h followed by wash-out for the indicated times. β-Actin was used as the loading control. The displayed image is representative of three biological replicates. (H) Immunoblot analysis (left) and quantification (right, n = 3) of BPTF in the total cell lysates. The lysates were isolated from Huh7 cells that were respectively pretreated with Poma (1 μM), MG132 (1 μM), and TP238 (10 μM) for 1 h before 8d treatment (10 μM for 24 h). β-Actin was used as the loading control. The displayed image is representative of three biological replicates. The data was presented as mean ± SD; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; Unpaired t test. (I) Immunoblotting of BPTF in the total cell lysates. The lysates were isolated from Huh7 cells that were treated with the deactivated degraders 16 , 17 and PROTAC degrader 8d (10 μM for 24 h), respectively. β-Actin was used as the loading control. The displayed image is representative of three biological replicates. (J) IP assay using anti-BPTF and IgG control to detect the endogenous association of BPTF with CRBN (E3 ligase) and the ubiquitination level of BPTF in Huh7 cells pretreated with MG132 (1 μM) for 3 h before 8d treatment (10 μM for 24 h). The displayed image is representative of three biological replicates.

Article Snippet: The primary antibodies used were BRD9 Rabbit mAb (CST, #58906), CECR2 Mouse mAb (Santa Cruz Biotechnology, sc-514878), BPTF Rabbit mAb (Abcam, ab72036), HPSE Mouse mAb (Santa Cruz Biotechnology, sc-515935), HS Mouse mAb (Millipore, #MAB2040), CRBN Polyclonal antibody (Proteintech, 28494-1-AP), anti-ubiquitin mouse mAb (NT) (PTM BIO, #PTM-5798), β-actin (8H10D10) Mouse mAb (Cell Signaling Technology, #3700).

Techniques: Western Blot, Isolation, Control, Expressing, Ubiquitin Proteomics