rabbit anti human leptin  (Santa Cruz Biotechnology)

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: A ntibodys and reagents 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: A ntibodys and reagents 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: A ntibodys and reagents 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

Journal: International journal of medical sciences

Article Title: Leptin regulates OPG and RANKL expression in Gingival Fibroblasts and Tissues of Chronic Periodontitis Patients.

doi: 10.7150/ijms.56151

Figure Lengend Snippet: Figure 1. Immunohistochemical staining of Leptin, OPG, and RANKL in control and chronic periodontitis groups. A-C. Immunohistochemical staining of Leptin in the control group(A), the moderate chronic periodontitis group (B), and the severe chronic periodontitis group (C). D-F. Immunohistochemical staining of OPG in the control group (D), the moderate chronic periodontitis group (E), and the severe chronic periodontitis group (F). G-I. Immunohistochemical staining of RANKL in the control group (G), the moderate chronic periodontitis group (H), and the severe chronic periodontitis group (I). Red arrows indicate the positive cells. EL: epithelial layer, LP: lamina propria.

Article Snippet: Leptin Rabbit Anti-Human Polyclonal Antibody (CSB-PA009805, CUSABIO, China), OPG Rabbit AntiHuman Polyclonal Antibody (CSB-PA003597, CUSABIO, China), RANKL Rabbit Anti-Human Polyclonal Antibody (CSB-PA023986LA01HU, CUSABIO, China), DAB (Servicebio, China), Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (Hyclone, America), Trypsin (Gibco, America), TRizol, PrimeScript RT reagent kit, SYBR Green mix (CWBIO, China) were used.

Techniques: Immunohistochemical staining, Staining, Control

Journal: International journal of medical sciences

Article Title: Leptin regulates OPG and RANKL expression in Gingival Fibroblasts and Tissues of Chronic Periodontitis Patients.

doi: 10.7150/ijms.56151

Figure Lengend Snippet: Figure 2. RANKL and OPG expression levels in leptin-treated HGF. A. Immunohistochemical staining of vimentin in HGF. B. Immunohistochemical staining of keratin in HGF. C. Expression of OPG, RANKL and OPG/RANKL at mRNA level in different leptin concentration groups (ug/ml).

Article Snippet: Leptin Rabbit Anti-Human Polyclonal Antibody (CSB-PA009805, CUSABIO, China), OPG Rabbit AntiHuman Polyclonal Antibody (CSB-PA003597, CUSABIO, China), RANKL Rabbit Anti-Human Polyclonal Antibody (CSB-PA023986LA01HU, CUSABIO, China), DAB (Servicebio, China), Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (Hyclone, America), Trypsin (Gibco, America), TRizol, PrimeScript RT reagent kit, SYBR Green mix (CWBIO, China) were used.

Techniques: Expressing, Immunohistochemical staining, Staining, Concentration Assay

Journal: Reproduction

Article Title: Crosstalk between estradiol and NFκB signaling pathways on placental leptin expression

doi: 10.1530/rep-20-0142

Figure Lengend Snippet: Figure 1 Inhibition of NFκB suppressed E2 effect on leptin expression BeWo cells (A) and (C) and term placental explants (B) and (D) were pretreated as indicated with sulfasalazine during 30 min, afterwards E2 was added. After 48 h (A) or 24 h (B), total RNA was extracted and Leptin mRNA expression was quantified by qRT-PCR. Cyclophilin was used as internal standard. Cell extracts (C) and placental explants extracts (D) were prepared as indicated in ‘Materials and methods’ section and proteins were separated on 12% SDS-PAGE gel. Leptin expression was determined by Western blot. Loading control was performed by immunoblotting the same membranes with anti-GAPDH. Standard protein markers were used to estimate the molecular weights. Molecular weight (kDa) is indicated at the right of each blot. Bands densitometry is shown in lower panels. Results are expressed as mean ± s.d. for ten independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs control and ##P < 0.01, ###P < 0.001 vs E2 treatment. Statistical analyses were performed by ANOVA followed by Bonferroni post hoc test.

Article Snippet: Membranes were equilibrated in 1× PBS and non-specific binding sites were blocked with 5% non-fat milk in PBS at room temperature for 1 h. Then they were immunoblotted with the specific polyclonal rabbit anti-human leptin Y20 (1:1000, Santa Cruz Biotechnology, Inc.), monoclonal mouse anti-p65 (1:1000, Santa Cruz Biotechnology, Inc.), or monoclonal mouse anti-pIκBα (1:1000 Santa Cruz Biotechnology, Inc.).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, SDS Page, Western Blot, Control, Molecular Weight

Journal: Reproduction

Article Title: Crosstalk between estradiol and NFκB signaling pathways on placental leptin expression

doi: 10.1530/rep-20-0142

Figure Lengend Snippet: Figure 2 Effect of p65 overexpression on E2 induced placental leptin expression. (A) BeWo cells were transiently transfected with pL1951 plasmid construction containing the promoter region of leptin gene from −1951 to +42 bp and different amounts of p65 expression plasmid. After transfection, BeWo cells were incubated for 48 h in DMEM-F12 media supplemented with 1% FBS and treated with E2 as indicated. (B) BeWo cells were transiently transfected with pL1951 plasmid construction, different amounts of plasmid expressing human

Article Snippet: Membranes were equilibrated in 1× PBS and non-specific binding sites were blocked with 5% non-fat milk in PBS at room temperature for 1 h. Then they were immunoblotted with the specific polyclonal rabbit anti-human leptin Y20 (1:1000, Santa Cruz Biotechnology, Inc.), monoclonal mouse anti-p65 (1:1000, Santa Cruz Biotechnology, Inc.), or monoclonal mouse anti-pIκBα (1:1000 Santa Cruz Biotechnology, Inc.).

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Incubation

Journal: Scientific Reports

Article Title: Abnormal Osteoblastic Response to Leptin in Patients with Adolescent Idiopathic Scoliosis

doi: 10.1038/s41598-019-53757-3

Figure Lengend Snippet: Leptin receptor expression level in control and AIS osteoblasts. ( A ) Representative Western blot was used to detect leptin receptors under basal and osteogenic conditions in control and AIS osteoblasts with transferrin receptor as loading control. ( B ) Corrected signal intensity of leptin receptor under basal and osteogenic conditions in control and AIS osteoblasts. A higher corrected signal intensity was observed in the AIS group at both conditions when compared with the controls. No significant difference was observed between the two conditions when compared within the AIS nor control groups.

Article Snippet: After blocking with 5% nonfat dry milk for 1 h at room temperature, membranes were incubated with rabbit IgG anti-human leptin receptor primary antibody (Santa Cruz, Dallas, USA) (1:500 in 3% BSA solution) or rabbit IgG anti-human transferrin receptor primary antibody (Cell Signaling Technology, Danvers, USA) (1:1000 in 3% BSA solution) overnight at 4 °C with agitation.

Techniques: Expressing, Control, Western Blot