pd l1 detection kit  (Elabscience Biotechnology)

Journal: Translational Oncology

Article Title: Activation of immune evasion machinery is a part of the process of malignant transformation of human cells

doi: 10.1016/j.tranon.2023.101805

Figure Lengend Snippet: Malignant transformation is associated with permanent activation of PI-3K/mTOR pathway and HIF-1/TGF-β axis. BEAS-2B cells were cultured for 10 days in the absence or presence of ptaquiloside containing bracken extracts (PTQL) followed by 14 days of culturing in PTQL-free medium. Upon completion of the whole process, we detected: (A) PI-3K activity and activating phosphorylation of mTOR, (B) mTOR-dependent phosphorylation of eIF4E-BP1, and (C) HIF-1 DNA-binding activity as well as the levels of secreted TGF-β. Images are from one experiment representative of 4 which gave similar results. Quantitative data are mean values ± SEM of 4 independent experiments.

Article Snippet: Mouse anti-Smad3 antibody, ELISA-based assay kits for the detection of VISTA, galectin-9, PD-L1 and TGF-β as well as mouse anti-Smad3 antibody were purchased from Bio-Techne (R&D Systems, Abingdon, UK).

Techniques: Transformation Assay, Activation Assay, Cell Culture, Activity Assay, Binding Assay

Journal: Translational Oncology

Article Title: Activation of immune evasion machinery is a part of the process of malignant transformation of human cells

doi: 10.1016/j.tranon.2023.101805

Figure Lengend Snippet: Malignant transformation leads to permanent upregulation of HIF-1α, TGF-β, galectin-9 and IDO1 mRNA levels in BEAS-2B cells. BEAS-2B cells were transformed as described above. After 5 th and 10 th days of culturing the cells in the absence or presence of PTQL and after day 14 of culturing them in PTQL-free medium we measured mRNA levels of HIF-1α, TGF-β, galectin-9 and IDO1. Data are mean values ± SEM of 4 independent experiments.

Article Snippet: Mouse anti-Smad3 antibody, ELISA-based assay kits for the detection of VISTA, galectin-9, PD-L1 and TGF-β as well as mouse anti-Smad3 antibody were purchased from Bio-Techne (R&D Systems, Abingdon, UK).

Techniques: Transformation Assay

Journal: Translational Oncology

Article Title: Activation of immune evasion machinery is a part of the process of malignant transformation of human cells

doi: 10.1016/j.tranon.2023.101805

Figure Lengend Snippet: Malignant transformation highly increases immune evasion potential of BEAS-2B and RC-124 cells. Non-transformed and PTQL-transformed BEAS-2B cells were co-cultured with TALL-104 cytotoxic T cells in the ratio 1:1 for 16 h. (A) Granzyme B protein levels (Western blot) and catalytic activity (biochemical assay) were measured as outlined in Materials and Methods. (B) Viabilities of all the cells co-cultured were measured using MTS assay. (C) Non-treated and PTQL-transformed BEAS-2B cells were co-cultured for 16 h in the ratio 1:1 with Jurkat T cells (CD4-positive helpers) or TALL-104 CD8-positive cytotoxic T cells followed by detection of secreted levels of galectin-9, TGF-β and IL-2 in the conditioned media. To investigate if Tim-3 or VISTA is used as a carrier protein in galectin-9 secretion process in BEAS-2B cells, normal and PTQL-transformed BEAS-2B cells were co-cultured with Jurkat T cells for 16 h. Then Tim-3 (D) or VISTA (E) were immunoprecipitated using ELISA plate (see Materials and Methods) and galectin-9 (if bound to Tim-3 or VISTA) was measured using Biotechne ELISA kit. (F) Galectin-9 was detectable when Tim-3 was immunoprecipitated and (G) undetectable on the immunoprecipitated VISTA. Normal and PTQL-treated RC-124 cells were co-cultured for 16 h with TALL-104 cells in the ratio 1:1. (H) PI-3K activity was measured using spectrophotometric assay and TGF-β release and phospho-S423/S425 Smad3 were measured by ELISA as outlined in Materials and Methods. (I) Galectin-9 release was measured by ELISA, and IDO1 activity as well as LKU release, were detected using colorimetric assays described in Materials and Methods. Images are from one experiment representative of 4 which gave similar results. Quantitative data are mean values ± SEM of 4 independent experiments.

Article Snippet: Mouse anti-Smad3 antibody, ELISA-based assay kits for the detection of VISTA, galectin-9, PD-L1 and TGF-β as well as mouse anti-Smad3 antibody were purchased from Bio-Techne (R&D Systems, Abingdon, UK).

Techniques: Transformation Assay, Cell Culture, Western Blot, Activity Assay, MTS Assay, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Spectrophotometric Assay