Journal: Cell Communication and Signaling : CCS

Article Title: Alpha5 nicotinic acetylcholine receptor mediated immune escape of lung adenocarcinoma via STAT3/Jab1-PD-L1 signalling

doi: 10.1186/s12964-022-00934-z

Figure Lengend Snippet: α5-nAChR mediates pSTAT3, Jab1 and PD-L1 expression in vitro. A , B Silencing α5-nAChR downregulated pSTAT3, Jab1 and PD-L1 expression in A549 cells and H1299 cells. C , D Nicotine promoted α5-nAChR, pSTAT3, Jab1 and PD-L1 expression in A549 cells and H1299 cells. Data are presented as the mean ± SEM of three independent experiments. ** P < 0.01, ns not significantly different

Article Snippet: Sections were blocked with normal serum at room temperature for 2 h. The samples on the slides were incubated at 4 °C overnight with a mouse monoclonal antibody against α5-nAChR (1:200, Proteintech, Cat. No. 66363-1-Ig), a mouse monoclonal antibody against PD-L1 (1:500, Proteintech, Cat. No. 66248-1-Ig), a rabbit monoclonal antibody against pSTAT3 (1:200, CST, Cat. No. 9145), a rabbit polyclonal antibody against Jab1 (1:500, Proteintech, Cat. No. 27511-1-AP), a rabbit monoclonal antibody against CD4 (1:1000, Abcam, Cat. No. ab183685), a rabbit monoclonal antibody against GB (1:3000, Abcam, Cat. No. ab255598).

Techniques: Expressing, In Vitro

Journal: Cell Communication and Signaling : CCS

Article Title: Alpha5 nicotinic acetylcholine receptor mediated immune escape of lung adenocarcinoma via STAT3/Jab1-PD-L1 signalling

doi: 10.1186/s12964-022-00934-z

Figure Lengend Snippet: α5-nAChR mediates PD-L1 expression via STAT3 binding to the PD-L1 or Jab1 promoter in LUAD cells. A , B pSTAT3 expression in the whole, nuclear, and cytoplasmic lysates of A549 cells and H1299 cells. C , D STAT3 bound to the PD-L1 and Jab1 promoter in A549 cells. E , F Silencing STAT3 downregulated PD-L1 and Jab1 expression in Α549 cells and H1299 cells. Data are presented as the mean ± SEM of three independent experiments. * P < 0.05, ** P < 0.01, ns not significantly different

Article Snippet: Sections were blocked with normal serum at room temperature for 2 h. The samples on the slides were incubated at 4 °C overnight with a mouse monoclonal antibody against α5-nAChR (1:200, Proteintech, Cat. No. 66363-1-Ig), a mouse monoclonal antibody against PD-L1 (1:500, Proteintech, Cat. No. 66248-1-Ig), a rabbit monoclonal antibody against pSTAT3 (1:200, CST, Cat. No. 9145), a rabbit polyclonal antibody against Jab1 (1:500, Proteintech, Cat. No. 27511-1-AP), a rabbit monoclonal antibody against CD4 (1:1000, Abcam, Cat. No. ab183685), a rabbit monoclonal antibody against GB (1:3000, Abcam, Cat. No. ab255598).

Techniques: Expressing, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Alpha5 nicotinic acetylcholine receptor mediated immune escape of lung adenocarcinoma via STAT3/Jab1-PD-L1 signalling

doi: 10.1186/s12964-022-00934-z

Figure Lengend Snippet: Jab1 stabilizes PD-L1 expression in LUAD cells. A PD-L1 expression in the si-NC or si-COPS5 groups treated with 50 μg/ml CHX. B PD-L1 expression in the si-NC or si-COPS5 groups treated with 10 μM MG-132 for 24 h. C Immunofluorescence staining of PD-L1 (green) and Jab1 (red). Scale bar, 100 μm. D The STAT3 binding site of the human Jab1 and PD-L1 promoter. Data are presented as the mean ± SEM of three independent experiments. ** P < 0.01

Article Snippet: Sections were blocked with normal serum at room temperature for 2 h. The samples on the slides were incubated at 4 °C overnight with a mouse monoclonal antibody against α5-nAChR (1:200, Proteintech, Cat. No. 66363-1-Ig), a mouse monoclonal antibody against PD-L1 (1:500, Proteintech, Cat. No. 66248-1-Ig), a rabbit monoclonal antibody against pSTAT3 (1:200, CST, Cat. No. 9145), a rabbit polyclonal antibody against Jab1 (1:500, Proteintech, Cat. No. 27511-1-AP), a rabbit monoclonal antibody against CD4 (1:1000, Abcam, Cat. No. ab183685), a rabbit monoclonal antibody against GB (1:3000, Abcam, Cat. No. ab255598).

Techniques: Expressing, Immunofluorescence, Staining, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Alpha5 nicotinic acetylcholine receptor mediated immune escape of lung adenocarcinoma via STAT3/Jab1-PD-L1 signalling

doi: 10.1186/s12964-022-00934-z

Figure Lengend Snippet: α5-nAChR, PD-L1, Jab1 and pSTAT3 expression in LUAD tumour xenograft tissues of nude mice. A α5-nAChR expression in NC, NC + nicotine, KD and KD + nicotine groups. B PD-L1 expression in NC, NC + nicotine, KD and KD + nicotine groups. C Jab1 expression in NC, NC + nicotine, KD and KD + nicotine groups. D pSTAT3 expression in NC, NC + nicotine, KD and KD + nicotine groups. Scare bar, 100 μm. Data are presented as the mean ± SEM of three independent experiments. * P < 0.05, ** P < 0.01

Article Snippet: Sections were blocked with normal serum at room temperature for 2 h. The samples on the slides were incubated at 4 °C overnight with a mouse monoclonal antibody against α5-nAChR (1:200, Proteintech, Cat. No. 66363-1-Ig), a mouse monoclonal antibody against PD-L1 (1:500, Proteintech, Cat. No. 66248-1-Ig), a rabbit monoclonal antibody against pSTAT3 (1:200, CST, Cat. No. 9145), a rabbit polyclonal antibody against Jab1 (1:500, Proteintech, Cat. No. 27511-1-AP), a rabbit monoclonal antibody against CD4 (1:1000, Abcam, Cat. No. ab183685), a rabbit monoclonal antibody against GB (1:3000, Abcam, Cat. No. ab255598).

Techniques: Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Alpha5 nicotinic acetylcholine receptor mediated immune escape of lung adenocarcinoma via STAT3/Jab1-PD-L1 signalling

doi: 10.1186/s12964-022-00934-z

Figure Lengend Snippet: Proposed signalling cascades of α5-nAChR and PD-L1 involves in immune escape in lung cancer. Nicotine binds and interacts with α5-nAChR on the cell surface, activates STAT3/PD-L1 or STAT3/Jab1-PD-L1 signalling, thereby α5-nAChR/PD-L1 axis mediated immune cells activity in lung carcinogenesis

Article Snippet: Sections were blocked with normal serum at room temperature for 2 h. The samples on the slides were incubated at 4 °C overnight with a mouse monoclonal antibody against α5-nAChR (1:200, Proteintech, Cat. No. 66363-1-Ig), a mouse monoclonal antibody against PD-L1 (1:500, Proteintech, Cat. No. 66248-1-Ig), a rabbit monoclonal antibody against pSTAT3 (1:200, CST, Cat. No. 9145), a rabbit polyclonal antibody against Jab1 (1:500, Proteintech, Cat. No. 27511-1-AP), a rabbit monoclonal antibody against CD4 (1:1000, Abcam, Cat. No. ab183685), a rabbit monoclonal antibody against GB (1:3000, Abcam, Cat. No. ab255598).

Techniques: Activity Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: HIC1 suppresses Tumor Progression and Enhances CD8 + T Cells Infiltration Through Promoting GSDMD-induced Pyroptosis in Gastric Cancer.

doi: 10.1002/advs.202412083

Figure Lengend Snippet: Figure 5. Jab1 specifically interacts with HIC1 and mediates the degradation of HIC1 in a ubiquitin-proteasome manner at K517. (A) Whole-cell extracts of MKN45 and HGC27 cells were isolated and subjected to a Co-IP assay to investigate the interaction between endogenous Jab1 and HIC1. (B) IF assay was employed to detect the co-localization of HIC1 and Jab1. (C) Western blot analysis revealed a significant reduction in HIC1 protein levels following transfection with Myc-Jab1 overexpression plasmid. (D) Cell lysates were collected for electrophoresis following treatment with MG132 (20 μm) for 6 h. (E,F) Western blot was subjected to explore the impact of Jab1 overexpression on the protein stability of HIC1 for MKN45 and HGC27 cells, as compared to an empty vector. CHX (50 μm). (G) Myc-Jab1, Flag-HIC1, and HA-ubiquitin plasmids were simultaneously transfected into cells and immunoprecipitated using HIC1 antibody prior to being immunoblotted. (H) Coomassie blue staining of the HIC1 protein complex with red arrows indicating the bands corresponding to HIC1 (76 kDa). (I) Ubiquitylation assays were carried out with FLAG-tagged wild-type HIC1 or lysine-to-arginine mutants K154R and K517R together with HA-Ub plasmid. (J) Alignment of the region corresponding to amino acids 515–525 of human HIC1 across different species. (K) GC cells transfected with the indicated plasmids were subjected to a ubiquitination experiment with the indicated antibodies.

Article Snippet: In Vitro Ubiquitination Assay: For in vitro ubiquitination assay, recombinant human Jab1 (Proteintech, Ag25956), ubiquitin (ABclonal, RPO1960), UBE1(R&D Systems, E-305-025), UBE2B (YEASEN, 20428es10) and ATP (Solarbio, C0550) were utilized.

Techniques: Ubiquitin Proteomics, Isolation, Co-Immunoprecipitation Assay, Western Blot, Transfection, Over Expression, Plasmid Preparation, Electrophoresis, Immunoprecipitation, Staining