Fig 1: TRIM46 interacts with TBK1. A. HEK293T cells were transfected with TRIM46-Myc plasmids with or not with TBK1-FLAG plasmids. After 24 h transfection, the cells were lysed and immuno-precipitated with anti-Myc antibody and subjected to western blotting with anti-Myc and anti-FLAG antibodies. The input cell lysates were analyzed by western blotting with anti-Myc and anti-FLAG antibodies, and GAPDH was used as an internal control. B. HEK293T cells were transfected with empty vector plasmids or TRIM46-Myc expression plasmids for 24 h, the cells were lysed and immuno-precipitated with anti-Flag antibody and subjected to western blotting with anti-Myc or anti-Flag antibodies. Whole cell lysates were subjected to western blotting with anti-Myc and anti-Flag antibodies and GAPDH was used as an internal control. All data were repeated for three independent experiments and the data shown as one representative of the triplicate experiments
Fig 2: TRIM46 promotes K48-linked ubiquitination of TBK1. HEK293T cells were transfected with indicated plasmids for 24 h, after transfection, the cells were lysed and immune-precipitated with anti-Flag antibody and subjected to western blotting for detection of WT-linked (A), K48-linked (B), K63-linked (C) ubiquitination. Anti-Myc, anti-Flag and GAPDH were used for the input
Fig 3: TRIM46 interacted with PHLPP2 and promoted PHLPP2 ubiquitination in LUAD cells.A Co-IP assay of TRIM46 and PHLPP2 in H358 cells. B Immunofluorescence assay of TRIM46 and PHLPP2 in H358 cells (scale bar: 20 μm). C qPCR and immunoblot analysis of PHLPP2 expression in both H358 cells and H1299 cells with TRIM46 silence. D Analysis of PHLPP2 expression in A549 cells transfected with WT-TRIM46, RING-mutant TRIM46, or vector. E IP analysis of the ubiquitination of PHLPP2 in A549 cells transfected with WT-TRIM46, RING-mutant TRIM46, or vector. F Immunohistochemical staining of TRIM46, PHLPP2, and p-AKT in cohort 1 (scale bar: 100 μm). G, H Statistical analysis of LUAD tissues under different staining conditions in cohort 1. Results were presented as the mean ± standard error (n = 3). Independent experiments were repeated three times.
Fig 4: Overexpression of TRIM46 promotes H7N9/ZJU-1 infection. A. A549 cells were transfected with lentivirus-mediated TRIM46-Myc plasmids or empty vector plasmids, after 72 h transfection, the cells were harvested and subjected to western blotting for TRIM46 overexpression analysis, GAPDH was used as an internal control. B. A549 cells transfected with lentivirus-mediated TRIM46-Myc plasmids or empty vector plasmids for 72 h, A549 cells were harvested and the relative levels of TRIM46 mRNA were analyzed by RT-qPCR. C. A549 cells were transfected with empty vector plasmids or TRIM46-Myc plasmids, after 72 h, A549 cells were infected with H7N9 /ZJU-1 (MOI = 1) or mock-treated for 12 h, the cells lysates were collected and subjected to western blotting with indicated antibodies. D. TRIM46-Myc overexpression A549 cells or empty vector-transfected A549 cells were infected with H7N9/ZJU-1 (MOI = 1) for 12 h, the relative levels of NP mRNA, cRNA and vRNA were analyzed by RT-qPCR. E. A549 cells were transfected with lentivirus-mediated TRIM46-Myc plasmids or empty vector plasmids, after 72 h transfection, the cells were infected with H7N9/ZJU-1 for 12 h, the supernatant was collected and the viral titers were determined by TCID50 method. The analysis results were presented with mean ± SD, in all situations, a p value < 0.05 was considered statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001
Fig 5: Depicting the possible molecular mechanisms by which TRIM46 regulates LUAD progression.TRIM46 activates AKT/HK2 signaling by modifying PHLPP2 ubiquitylation to promote glycolysis and chemoresistance of lung cancer cells.
Supplier Page from Abcam for Anti-TRIM46 antibody