antibodies against lepr  (R&D Systems)

Journal: bioRxiv

Article Title: Quantitative Multicolored Deep Imaging of Human Bones Reveals a Composite Osteo-Sinusoidal Niche for Mesenchymal Stromal Cells

doi: 10.1101/2025.10.07.680053

Figure Lengend Snippet: a,b , Colocalization of LEPR protein (antibody staining, pseudocoloured yellow) and CXCL12 mRNA (HCR RNA-FISH, red) in representative 2D optical sections from ( a ) a 3D light-sheet scan and ( b ) a confocal scan of tissue-cleared human bone. Left panels show LEPR omitted; right panels include all channels. Nuclei (YOPRO, green) and bone matrix (autofluorescence at 405 nm, cyan) were visualized in ( a ) but not ( b ) due to sample bleaching. c , Confocal image of a 6-μm thick section from decalcified human bone stained for LEPR (green), CXCL12 mRNA (red), and counterstained with DAPI (blue). Single and merged channels are shown. d–f , Nuclear staining of aged human bone with YOPRO1 (green). d, Raw YOPRO+ signals in a light-sheet scan. e, YOPRO+ nuclei overlaid with cell positions detected by the Imaris Spot function. f, Optical sections showing overlay of YOPRO+ nuclei with segmented cell masks (grey circles). g–l , Detection of nucleated CXCL12+ cells in aged bone. g–i, Representative 2D optical sections from a 3D light-sheet scan showing CXCL12 mRNA (HCR, red), CD31+ vessels (yellow), and YOPRO1+ nuclei (green). CXCL12 signal is omitted in g and present in h ; i , colocalized CXCL12+YOPRO+ cells detected by Imaris Spot function are marked by grey circles. j–l , 3D projections of the same region as a raw scan ( j ) and detected double-positive cells at low ( k ) and high ( l ) magnification, with double-positive cells represented as green spheres. j , CD31 and YOPRO channels only; k,l , all channels including CXCL12. All images are cropped from large tissue volumes (∼2 × 2 × 2 mm) scanned by light-sheet microscopy.

Article Snippet: Following HCR, tissue sections were blocked using the same staining buffer as used in DeepBone, then incubated with primary antibodies against LEPR (conjugated to AlexaFluor488; #MAB867, R&D Systems) or Ki67 (conjugated to AlexaFluor546; SolA15, Thermofisher).

Techniques: Staining, Microscopy

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: EREG mediates glucose uptake via PI3K with transient activation of ERK. ( A ) FD-glucose uptake in 3T3-L3 preadipocytes treated with or without EREG (50 ng/mL) and in the presence of inhibitors for EGFR-I (AG1478, 10 µM), EGFR and ErbB2 (AST-1306 or CI-1033 10 µM), dual IR/IGF-1R inhibitor (BMS 536924, 1 µM), and SRC-I, AZM475271, 1 µM) for 30 min. Cells were starved for 90 min before stimulation. Dashed line shows glucose uptake in the presence of insulin (Ins, 10 µg/mL). ( B ) FD-glucose uptake was measured in mouse 3T3-L1 preadipocytes with or without EREG (50 ng/mL) and inhibitors of MEK1/2 and PI3K (MEK1/2-I, U0126 10 μM, and PI3K-I, wortmannin 200 nM). Data (mean ± SD, n = 6) are shown as a percentage of control (Veh 100%). Unpaired Student’s t -test. ( C ) 3T3-L1 preadipocytes were stimulated with EREG at different concentrations (0–100 ng/mL) for 5 or 15 min. The total and phosphorylated levels of AKT, STAT3, STAT5, and ERK were measured by Western blot in duplicates. Data are shown in a representative Western blot. ( D ) The kinetics of pERK expression was quantified based on the Western blots. pAKT, p-STAT3, and p-STAT5 analysis are described in . Pearson correlation analysis. ( E ) 3T3-L1 preadipocytes were stimulated with or without EREG or EGF (50 ng/mL, each) for 30 min in the presence and absence of EGFR inhibitor AST1306 (100 nM), and antibody against mouse LepR (Invitrogen, PA1-053, 10 μg/mL). For inhibition, cells were pre-treated 30 min before EREG and EGF stimulation. ( F ) FD-glucose uptake was measured in mouse 3T3-L3 preadipocytes pre-treated with either Veh (DMSO) or ERK inhibitors (U0126, SCH772984, or DEL 22379, each 10 µM in DMSO) for 40 min. Then, cells were treated with either Veh (PBS), mouse EREG (50 ng/mL), or mouse leptin (Lep, 200 ng/mL) for 80 min. Data are shown as a percentage of Veh-treated control (100%, n = 7 per group). Unpaired Student’s t -test. ns , not significant ( p > 0.05).

Article Snippet: The antibody against LepR and β-actin were acquired from ThermoFisher Scientific (PA1-28844, LOT# SH2429627A) and Sigma-Aldrich (A5441).

Techniques: Activation Assay, Western Blot, Expressing, Inhibition

Journal: American Journal of Translational Research

Article Title: Effects of the multifunctional hormone leptin on orthodontic tooth movement in rats

doi:

Figure Lengend Snippet: The RANKL expression was downregulation by leptin in human periodontal ligament cells with force application. A. Schema of the in-vitro compression force application to hPDLCs. B. Immunofluorescence staining showed that showed that number of the RANKL positive cells was reduced by leptin administration and reversed by LepR siRNA transfection. C. Quantitative PCR results showed that the expression of RANKL mRNA was significantly elevated after compression force application and reduced by leptin administration which was reversed by LepR siRNA transfection. D. Quantitative PCR results showed that administration of leptin to hPDLCs was able to significantly reduce RANKL/OPG ratio under compression which was reversed by LepR transfection. E. Quantitative PCR results showed that the expression of PPAR-γ mRNA was significantly inhibited by leptin which was reversed by LepR siRNA transfection. F. Quantitative PCR results showed that the expression of GSK-3β mRNA was significantly inhibited by leptin which was reversed by LepR siRNA transfection. **denotes significant difference, P<0.01; *denotes significant difference, P<0.05; NS denotes no statistical significance; scale bar: 20 μm.

Article Snippet: Cells were incubated with primary antibodies against LepR or RANKL (LepR, 1:100, RANKL, 1:100, Santa Cruz, USA) to detect the leptin receptor or RANKL expression on PDLCs and then washed, incubated with FITC-conjugated secondary antibodies (Zhongshan Golden Bridge Biotechnology, Beijing, China).

Techniques: Expressing, In Vitro, Immunofluorescence, Staining, Transfection, Real-time Polymerase Chain Reaction