cpsf1 3  (Bethyl)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: ABL kinases regulate the stabilization of HIF-1α and MYC through CPSF1.

doi: 10.1073/pnas.2210418120

Figure Lengend Snippet: Fig. 4. CPSF1 is a HIF-1α-targeting E3-ligase. (A) Scheme of the FACS-based CRISPR E3 ligase screen performed. (B) Snake plot depicting the β scores across 593 E3-ligase-related gene identified in PC9 lung cancer cells treated with ABL kinase inhibitor under hypoxia. (C) IB analysis of IP- and WCL-derived HEK293T cells transfected with 3xFlag-HIF1A and HA-CPSF1 constructs for 24 h and then treated with MG132 during hypoxia for 24 h before harvesting. (D) IB analysis of WCL derived from HEK293T cells transfected with 3xFlag-HIF1A and HA-CPS1 constructs for 24 h and then exposed to normoxia or hypoxia for 24 h. (E) IB analysis of WCL derived from HEK293T cells transfected with 3xFlag-HIF1A and HA-CPSF1 constructs for 24 h and then treated with or without MG132 during hypoxia for 24 h before harvesting. (F) IB analysis of IP derived from HEK293T cells transfected with 3xFlag-HIF1A and HA-CSPF1 constructs for 24 h and then treated with MG132 for the final 24 h. (G) Proposed model depicting CPSF1-dependent HIF-1α ubiquitination and proteasome-mediated protein degradation.

Article Snippet: The following primary antibodies were used for IB analyses: ABL1 (Sigma MAB1130 1:1,000), ABL2 (Abnova, H00000027-M03, 1:500), ACTIN (Cell Signaling 3700 1:10,000), CPSF1 (Bethyl A301-580A 1:1,000), CRKL (Santa Cruz sc-319 1:1,000), CRKL p-Y207 (Cell Signaling 3181 1:500), Flag (Sigma M2 1:5,000), GFP (Cell Signaling 2956 1:1,000), HA (Cell Signaling 2367 1:1,000), HIF-1α (BD Bioscience 610959 1:1,000), HIF-2α (Novus NB100-122 1:1,000), HIF-1β (Novus NB100-124 1:1,000), MYC (Cell Signaling 5605 1:5,000), Ubb (Santa Cruz sc-166553 1:1,000), and p-Y 4G10 (Sigma 05-321).

Techniques: CRISPR, Derivative Assay, Transfection, Construct, Ubiquitin Proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: ABL kinases regulate the stabilization of HIF-1α and MYC through CPSF1.

doi: 10.1073/pnas.2210418120

Figure Lengend Snippet: Fig. 5. CPSF1 is an E3-ligase targeting the HIF-1α DNA-binding domain. (A, Top) Structural diagram of full-length HIF-1α and truncation mutants. (A, Bottom) IB analysis of WCL derived from HEK293T cells transfected with HA-CPSF1 and indicated 3xFlag-HIF1A truncation constructs for 24 h and exposed to hypoxia for 24 h. (B) HDOCK protein–protein docking server prediction of the interaction of full-length CPSF1 (PDB: 6F9N) with HIF-1α AA 1-71 (PDB:4ZPR). CPSF1 is shown in orange and HIF-1α is shown in blue. (C) IB analysis of WCL derived from HEK293T cells transfected with indicated 3xFlag-HIF1A and HA-CPSF1 constructs for 24 h and exposed to hypoxia for 24 h. (D) IB and IP analysis of WCL derived from HEK293T cells transfected with HA-CPSF1 and indicated 3xFlag-HIF1A constructs for 24 h and exposed to hypoxia for 24 h. (E) IB analysis of WCL-derived 293T cells transfected with indicated 3xFlag-HIF1A constructs for 24 h and then treated with or without ABL001 during hypoxia for 24 h. (F) IB analysis of WCL derived from HEK293T cells transfected with 3xFlag-HIF1A and indicated HA-CPSF1 constructs for 24 h and exposed to hypoxia for 24 h. (G) Scheme of CPSF1- and VHL-resistant HIF-1α mutants. (H) IB analysis of WCL derived from HEK293T cells transfected with indicated 3xFlag-HIF1A and HA-VHL constructs for 24 h and exposed to hypoxia for 24 h. (I) IB analysis of WCL derived from HEK293T cells transfected with indicated 3xFlag-HIF1A and HA-CPSF1 constructs for 24 h and exposed to hypoxia for 24 h. (J) IB analysis of WCL-derived 293T cells transfected with indicated 3xFlag-HIF1A constructs for 24 h and then treated with or without ABL001 during hypoxia for 24 h.

Article Snippet: The following primary antibodies were used for IB analyses: ABL1 (Sigma MAB1130 1:1,000), ABL2 (Abnova, H00000027-M03, 1:500), ACTIN (Cell Signaling 3700 1:10,000), CPSF1 (Bethyl A301-580A 1:1,000), CRKL (Santa Cruz sc-319 1:1,000), CRKL p-Y207 (Cell Signaling 3181 1:500), Flag (Sigma M2 1:5,000), GFP (Cell Signaling 2956 1:1,000), HA (Cell Signaling 2367 1:1,000), HIF-1α (BD Bioscience 610959 1:1,000), HIF-2α (Novus NB100-122 1:1,000), HIF-1β (Novus NB100-124 1:1,000), MYC (Cell Signaling 5605 1:5,000), Ubb (Santa Cruz sc-166553 1:1,000), and p-Y 4G10 (Sigma 05-321).

Techniques: Binding Assay, Derivative Assay, Transfection, Construct

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: ABL kinases regulate the stabilization of HIF-1α and MYC through CPSF1.

doi: 10.1073/pnas.2210418120

Figure Lengend Snippet: Fig. 6. ABL kinases protect HIF-1α from CPSF1-dependent degradation. (A) IB analysis of WCL derived from HEK293T and PC9 cells transduced with indicated gRNA/CRISPR- Cas9 constructs and then treated with ABL001 during hypoxia for 24 h. (B) IB analysis of WCL derived from HEK293T cells transfected with 3xFlag-HIF1A, ABL1-eGFP, and HA-CPSF1 constructs for 24 h and then exposed to hypoxia for 24 h. (C) IB and IP analysis of WCL derived from PC9 cells treated with ABL001 (24 h) and MG132 (final 6 h) during hypoxia for 24 h. IgG sample added to the vehicle and MG132-treated lysate. (D) Proposed model depicting loss of ABL kinase activity leading to CPSF1-dependent HIF-1α ubiquitination and proteasome-mediated protein degradation.

Article Snippet: The following primary antibodies were used for IB analyses: ABL1 (Sigma MAB1130 1:1,000), ABL2 (Abnova, H00000027-M03, 1:500), ACTIN (Cell Signaling 3700 1:10,000), CPSF1 (Bethyl A301-580A 1:1,000), CRKL (Santa Cruz sc-319 1:1,000), CRKL p-Y207 (Cell Signaling 3181 1:500), Flag (Sigma M2 1:5,000), GFP (Cell Signaling 2956 1:1,000), HA (Cell Signaling 2367 1:1,000), HIF-1α (BD Bioscience 610959 1:1,000), HIF-2α (Novus NB100-122 1:1,000), HIF-1β (Novus NB100-124 1:1,000), MYC (Cell Signaling 5605 1:5,000), Ubb (Santa Cruz sc-166553 1:1,000), and p-Y 4G10 (Sigma 05-321).

Techniques: Derivative Assay, Transduction, CRISPR, Construct, Transfection, Activity Assay, Ubiquitin Proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: ABL kinases regulate the stabilization of HIF-1α and MYC through CPSF1.

doi: 10.1073/pnas.2210418120

Figure Lengend Snippet: Fig. 9. ABL kinase protects MYC from CPSF1-dependent degradation. (A) IB analysis of IP- and WCL-derived HEK293T cells transfected with Flag-MYC and HA-CPSF1 constructs for 48 h. (B) IB analysis of WCL derived from HEK293T cells transfected with Flag-MYC and HA-CPSF1 constructs for 48 h. (C) IB analysis of WCL derived from HEK293T cells transfected with Flag-MYC and HA-CPSF1 constructs for 24 h and then treated with MG132 for 24 h before harvesting during normoxia. (D, Top) Structural diagram of full-length MYC and truncation mutants. (A, Bottom) IB analysis of WCL derived from HEK293T cells transfected with HA-CPSF1 and indicated Flag-MYC truncation constructs for 24 h and exposed to hypoxia for 24 h. (E) IB analysis of WCL derived from HEK293T cells transfected with Flag-MYC and ABL1-eGFP constructs for 48 h during normoxia. (F) IB analysis of WCL derived from HEK293T cells transfected with Flag-MYC for 24 h and then treated with ABL001 and GNF5 as indicated for 24 h during normoxia. (G and H) IB analysis of WCL derived from PC9 cells treated with ABL001, GNF5, and MG132 as indicated for 24 h during normoxia. (I) IB analysis of WCL derived from HEK293T cells transfected with Flag-MYC, ABL1-eGFP, and HA-CPSF1 constructs for 48 h. (J) Model for the ABL kinase regulation of HIF-1α and MYC through CPSF1.

Article Snippet: The following primary antibodies were used for IB analyses: ABL1 (Sigma MAB1130 1:1,000), ABL2 (Abnova, H00000027-M03, 1:500), ACTIN (Cell Signaling 3700 1:10,000), CPSF1 (Bethyl A301-580A 1:1,000), CRKL (Santa Cruz sc-319 1:1,000), CRKL p-Y207 (Cell Signaling 3181 1:500), Flag (Sigma M2 1:5,000), GFP (Cell Signaling 2956 1:1,000), HA (Cell Signaling 2367 1:1,000), HIF-1α (BD Bioscience 610959 1:1,000), HIF-2α (Novus NB100-122 1:1,000), HIF-1β (Novus NB100-124 1:1,000), MYC (Cell Signaling 5605 1:5,000), Ubb (Santa Cruz sc-166553 1:1,000), and p-Y 4G10 (Sigma 05-321).

Techniques: Derivative Assay, Transfection, Construct