Journal: Kidney international

Article Title: Gluten exacerbates IgA nephropathy in humanized mice through gliadin-CD89 interaction.

doi: 10.1038/ki.2015.94

Figure Lengend Snippet: Figure 4| Gliadin directly interacts with sCD89. (a) Binding of sCD89 to gliadin, compared with blank and human serum albumin used as negative controls, as detected by ELISA using biotinylated sCD89. (b) Dose-dependent binding of sCD89 to gliadin. n = 3 experiments. ELISA, enzyme-linked immunosorbent assay.

Article Snippet: Plates were coated overnight with indicated antibodies or proteins (10 μg/ml) and incubated with samples as described.7 After washing, the anti-human IgA mAb coupled with the alkaline phosphatase (BD Biosciences) was added for 1 h. For binding assays, biotinylated sCD89, or biotinylated anti-ovalbumin α1-KI IgA1, or biotinylated human IgA (MP Biomedicals, Illkirch-Graffenstaden, France) was incubated for 2 h, and after washing streptavidin-alkaline phosphatase (Jackson Laboratories, Suffolk, UK) was added for 30 min.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Annals of Translational Medicine

Article Title: Autocrined leptin promotes proliferation of non-small cell lung cancer (NSCLC) via PI3K/AKT and p53 pathways

doi: 10.21037/atm-20-7482

Figure Lengend Snippet: Representative image of leptin expression in lung adenocarcinoma. Leptin expression in tumors and paired normal lung tissues was detected by immunochemical staining analysis. (A) Leptin protein was overexpressed in a moderately differentiated lung adenocarcinoma samples with acinar predominant growth compared with normal control lung tissue. It can be seen that leptin protein was mainly expressed in the parenchyma (epithelial cells) of lung cancer tissues. (B) There was no difference in the expression of leptin protein in a case of well-differentiated lung adenocarcinoma with lepidic predominant (LPA) growth compared with normal control lung tissue. Leptin was seen to be expressed at low levels in the parenchyma of lung cancer tissue and in normal lung tissue. (C) Leptin protein was overexpressed in a highly differentiated lung adenocarcinoma sample with papillary predominant growth (PPA) compared to normal control lung tissue. It can be seen that leptin protein was mainly expressed in the parenchyma of lung cancer tissues. (D) Leptin protein was overexpressed in a poorly differentiated lung adenocarcinoma sample with micropapillary predominant growth (MPA) compared to normal control lung tissue. It can be seen that leptin protein was expressed in both parenchyma and mesenchyme of lung cancer tissues. (E) Leptin protein was overexpressed in a highly differentiated lung adenocarcinoma with solid predominant growth compared to normal control lung tissue. It can be seen that leptin protein was expressed in the parenchyma, mesenchyme, and normal control lung tissues of lung cancer, but the expression level was low in normal tissues. (F) Leptin protein was overexpressed in a lung minimally invasive adenocarcinoma (MIA) compared to normal control lung tissue from the same patient. All experiments were repeated three times. Immunohistochemistry staining IHC staining of leptin was performed according to the manufacturer’s instructions. A streptavidin-peroxidase staining kit was purchased from ZSGB BIO (Beijing, China). The paraffin-embedded tissues were prepared by a pathology specialist, and dewaxed and rehydrated in the lab. The experimental steps were carried out according to the instructions of the SP kit. The tissues were incubated with primary antibodies diluted to the recommended concentration overnight at 4 °C with antibodies that was dilute to the recommended concentration. Then 3,3'-diaminobenzidine (DAB) staining was performed, and the results were observed under a microscope.

Article Snippet: Rabbit anti-human leptin monoclonal antibody was purchased from Bioss Antibodies (Woburn, MA, USA); rabbit anti-human LC3, β-actin, and P62 monoclonal antibodies were purchased from Proteintech; rabbit anti-human Ob-R monoclonal antibodies were purchased from Shenyang Wanlei Biological Company (Shenyang, China); Rabbit anti-human AKT , p - AKT , p - ERK , p - NF -κ B , p65 , cyclin D1 , CDK2 , p53 , MDM2 , p - MDM2 , and p-mTOR monoclonal antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Staining, Immunohistochemistry, Incubation, Concentration Assay, Microscopy

Journal: Annals of Translational Medicine

Article Title: Autocrined leptin promotes proliferation of non-small cell lung cancer (NSCLC) via PI3K/AKT and p53 pathways

doi: 10.21037/atm-20-7482

Figure Lengend Snippet: The effect of endogenous leptin expression on the proliferation of pulmonary adenocarcinoma cells. (A) The clone formation test of H1299 and A549 cells on the effect of proliferation of pulmonary adenocarcinoma; 50 ng/mL artificial recombinant leptin protein was used to simulate the impact of exogenous leptin in circulating blood on tumor cells; (B) the flow cell cycle analysis of PI staining showing the effect of endogenous leptin expression on the cell cycle of lung adenocarcinoma in H1299 and A549 cells; (C) AV/PI double-staining flow cytometry apoptotic cell test showing the effect of endogenous leptin expression in the apoptosis of lung adenocarcinoma in H1299-sh and A549 cells. All the experiments were repeated three times. Immunohistochemistry staining IHC staining of leptin was performed according to the manufacturer’s instructions. A streptavidin–peroxidase staining kit was purchased from ZSGB BIO (Beijing, China). The paraffin-embedded tissues were prepared by a pathology specialist, and dewaxed and rehydrated in the lab. The experimental steps were carried out according to the instructions of the SP kit. The tissues were incubated with primary antibodies diluted to the recommended concentration overnight at 4 °C with antibodies that was dilute to the recommended concentration. Then 3,3'-diaminobenzidine (DAB) staining was performed, and the results were observed under a microscope (100×).

Article Snippet: Rabbit anti-human leptin monoclonal antibody was purchased from Bioss Antibodies (Woburn, MA, USA); rabbit anti-human LC3, β-actin, and P62 monoclonal antibodies were purchased from Proteintech; rabbit anti-human Ob-R monoclonal antibodies were purchased from Shenyang Wanlei Biological Company (Shenyang, China); Rabbit anti-human AKT , p - AKT , p - ERK , p - NF -κ B , p65 , cyclin D1 , CDK2 , p53 , MDM2 , p - MDM2 , and p-mTOR monoclonal antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Recombinant, Cell Cycle Assay, Staining, Double Staining, Flow Cytometry, Immunohistochemistry, Incubation, Concentration Assay, Microscopy

Journal: Annals of Translational Medicine

Article Title: Autocrined leptin promotes proliferation of non-small cell lung cancer (NSCLC) via PI3K/AKT and p53 pathways

doi: 10.21037/atm-20-7482

Figure Lengend Snippet: Molecular mechanisms of endogenous leptin expression effect on the proliferative ability of lung adenocarcinoma cells. (A) Effect of endogenous leptin expression on key signaling molecules of PI3K/AKT pathway and its downstream signaling pathway in H1299 and A549 cells detected by western blotting; (B) effect of endogenous leptin expression on the p53 signaling pathway in A549 cells by western blotting; (C) endogenous leptin expression level on the expression level of autophagy-related protein LC3-II in H1299 cell lines detected by protein immunofluorescence. (D) Effect of endogenous leptin expression on key signaling molecules of the mTOR pathway and its downstream signalings in H1299 and A549 cells detected by western blotting. All the experiments were repeated three times. Scale bar: 50 µm.

Article Snippet: Rabbit anti-human leptin monoclonal antibody was purchased from Bioss Antibodies (Woburn, MA, USA); rabbit anti-human LC3, β-actin, and P62 monoclonal antibodies were purchased from Proteintech; rabbit anti-human Ob-R monoclonal antibodies were purchased from Shenyang Wanlei Biological Company (Shenyang, China); Rabbit anti-human AKT , p - AKT , p - ERK , p - NF -κ B , p65 , cyclin D1 , CDK2 , p53 , MDM2 , p - MDM2 , and p-mTOR monoclonal antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Western Blot, Immunofluorescence