anti stat1 p84 91 c 136  (Santa Cruz Biotechnology)

Journal: Theranostics

Article Title: UBR5 promotes tumor immune evasion through enhancing IFN-γ-induced PDL1 transcription in triple negative breast cancer

doi: 10.7150/thno.74989

Figure Lengend Snippet: UBR5 is crucial for IFN-γ-induced activation of STAT1 and IRF1 transcription. (A-B) The mRNA (A) and protein (B) levels of UBR5, STAT1, pSTAT1 and IRF1 were detected in WT, Ubr5 -/- and h UBR5 -reconstituted Ubr5 -/- 4T1 cells with or without IFN-γ stimulation. (C-D) The mRNA (C) and protein levels (D) of STAT1 and IRF1were detected in IFN-γ-treated UBR5-knockdown BT549 (C and D) and MDA-MB-231 (D) stable cell lines. (E-G) BT549, MDA-MB-231 and MCF7 cells were transfected with either an empty vector or UBR5 plasmids. 24 hours later, the cells were treated with IFN-γ for 24 h. Then the mRNA and protein levels of STAT1 (E and G) and IRF1 (F and G) were measured by qPCR and western blot, respectively. (H) Surface PD-L1 levels were detected in IFN-γ-treated GFP, Ubr5 -/- 4T1 cells and GFP 4T1 cells treated with siNC, siSTAT1 or siIRF1. GAPDH was used for normalization. (I) Luciferase reporter vectors containing either STAT1 or IRF1 promoter regions were cotransfected with an empty vector or UBR5 plasmids into the indicated cells. After 24 h, the transfected cells were treated with IFN-γ stimulation for 24 h, the cells were lysed to perform luciferase assay. The results are presented as the mean ± SEM from three individual experiments. *P < 0.05, **P < 0.01, ***P< 0.001, ****P < 0.0001. (J) The IRF1 and STAT1 binding sites in the m Pdl1 promoter region were predicted using the ALGGEN website. (K) Summary of the results of a ChIP assay using anti-IRF1, STAT1, H3K4me1 and H3K27ac antibodies in WT, Ubr5 -/- or h UBR5 -reconstituted Ubr5 -/- 4T1 cells after treatment with or without IFN-γ.

Article Snippet: Proteins were resolved on a 10% SDS PAGE gel and transferred to the NC membrane, blocked with 5% milk and probed for monoclonal antibodies against UBR5 (Santa Cruz, sc-515494), PD-L1 (Proteintech) #66248-1-lg, EIF2AK2 (Beyotime) #AF2125, STAT1 p84/91 (C-136) (Santa Cruz, sc-464), STAT1 (D1K9Y) Rabbit (Cell Signal Technology) mAb #14994, Phospho-STAT1 (Tyr701) Rabbit (Beyotime) #AF5935, IRF1 (E-4) (Santa Cruz, sc-514544) or anti-GAPDH (Proteintech).

Techniques: Activation Assay, Knockdown, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Luciferase, Binding Assay

Journal: Theranostics

Article Title: UBR5 promotes tumor immune evasion through enhancing IFN-γ-induced PDL1 transcription in triple negative breast cancer

doi: 10.7150/thno.74989

Figure Lengend Snippet: UBR5-mediated transactivation of PDL1 is independent of its E3 ubiquitin ligase. (A-D) MDA-MB-231 cells were cotransfected with WT, HECT-mutation (UBR5-C2768A) or PABC-deletion UBR5 (A) together with luciferase reporters containing the human PDL1 WT promoter or a promoter with 2 deletions, including the STAT1/3 or IRF1 binding sites (B), and then stimulated with IFN-γ for 24 h. Luciferase activity was measured in cell lysates of MDA-MB-231 (C) and 4T1 cells (D) by dual luciferase assay. (E-F) The protein levels of UBR5 (E) and PD-L1 (F) were measured in IFN-γ-treated GFP, Ubr5 -/- , h UBR5 -reconstituted Ubr5 -/- 4T1 cells and Ubr5 -/- 4T1 cells reconstituted with either h UBR5 -C2768 or h UBR5 -ΔPABC. The results are presented as the mean ± SEM from three individual experiments. ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Proteins were resolved on a 10% SDS PAGE gel and transferred to the NC membrane, blocked with 5% milk and probed for monoclonal antibodies against UBR5 (Santa Cruz, sc-515494), PD-L1 (Proteintech) #66248-1-lg, EIF2AK2 (Beyotime) #AF2125, STAT1 p84/91 (C-136) (Santa Cruz, sc-464), STAT1 (D1K9Y) Rabbit (Cell Signal Technology) mAb #14994, Phospho-STAT1 (Tyr701) Rabbit (Beyotime) #AF5935, IRF1 (E-4) (Santa Cruz, sc-514544) or anti-GAPDH (Proteintech).

Techniques: Ubiquitin Proteomics, Mutagenesis, Luciferase, Binding Assay, Activity Assay

Journal: Theranostics

Article Title: UBR5 promotes tumor immune evasion through enhancing IFN-γ-induced PDL1 transcription in triple negative breast cancer

doi: 10.7150/thno.74989

Figure Lengend Snippet: The transactivation of UBR5 to STAT1 and PDL1 is mediated by protein kinase RNA-activated. (A-B) The mRNA (A) and protein levels (B) of EIF2AK2 in WT, Ubr5 -/- , h UBR5 -reconstituted Ubr5 -/- 4T1 cells and Ubr5 -/- 4T1 cells reconstituted with either h UBR5 -C2768A or h UBR5 -ΔPABC were measured by qPCR and western blot analysis after cells were treated with or without IFN-γ. (C-D) The mRNA (C) and protein levels (D) levels of EIF2AK2 in WT and UBR5-knockdown BT549 cells were measured by qPCR and western blot analysis after cells were treated with IFN-γ for 24 h. (E-G) The mRNA (E) and protein levels of UBR5, STAT1, IRF1 and EIF2AK2 (F) and relative fold changes of cell-surface PD-L1 levels (G) in IFN-γ-treated WT, Ubr5 -/- , and 4T1 cells with stable knockdown by shEif2ak2 #1 and shEif2ak2 #1. Shscramble served as the control. (H-J) The mRNA levels (H) of Eif2ak2 , Ubr5 , Pdl1 , Stat1 and Irf1 , the protein levels of UBR5, EIF2AK2, STAT1 and IRF1 (I) and the surface levels of PD-L1 (J) were measured in IFN-γ-treated 4T1 WT, Ubr5 -/- and m Eif2ak2 -reconstituted Ubr5 -/- cell lines. The results are presented as the mean ± SEM from three individual experiments. ns, no significance, *P< 0.05, **P < 0.01, ***P< 0.001, ****P < 0.0001. (K) The correlations of EIF2AK2 with UBR5 , PDL1 and STAT1 mRNA levels were assessed in the TCGA BRCA database (1104 samples) by starBase database. (L) The summary correlations of EIF2AK2 with UBR5 , PDL1 , STAT1 and IRF1 mRNA levels (normalizated to GAPDH) were analysed in the TCGA BRCA database by starBase and GEPIA database respectively.

Article Snippet: Proteins were resolved on a 10% SDS PAGE gel and transferred to the NC membrane, blocked with 5% milk and probed for monoclonal antibodies against UBR5 (Santa Cruz, sc-515494), PD-L1 (Proteintech) #66248-1-lg, EIF2AK2 (Beyotime) #AF2125, STAT1 p84/91 (C-136) (Santa Cruz, sc-464), STAT1 (D1K9Y) Rabbit (Cell Signal Technology) mAb #14994, Phospho-STAT1 (Tyr701) Rabbit (Beyotime) #AF5935, IRF1 (E-4) (Santa Cruz, sc-514544) or anti-GAPDH (Proteintech).

Techniques: Western Blot, Knockdown, Control