human leptin receptor polyclonal antibody  (MyBiosource Biotechnology)

Journal: Annals of Clinical and Translational Neurology

Article Title: Obesity and the risk of Multiple Sclerosis. The role of Leptin

doi: 10.1002/acn3.51291

Figure Lengend Snippet: (A) Body mass index (BMI) positively correlated with serum leptin levels. (B) Serum leptin levels were higher in obese patients compared to overweight and normal/underweight patients. Similarly, overweight patients showed increased serum leptin levels compared to normal/underweight patients. No statistically significant differences were found when MS and control patients were compared in each subgroup. Each circle represents values from a single individual. Data are presented as mean ± SEM. Kruskal–Wallis test of one‐way ANOVA and post hoc data analysis applying Dunn's multiple comparison test, performed to analyze differences between groups. ** P < 0.01, **** P < 0.0001.

Article Snippet: For blocking experiments human leptin receptor polyclonal antibody (MyBioSource, San Diego CA) was used at a final concentration of 20 μ g/mL; control was an irrelevant isotype‐matched antibody (R&D Systems).

Techniques: Control, Comparison

Journal: Annals of Clinical and Translational Neurology

Article Title: Obesity and the risk of Multiple Sclerosis. The role of Leptin

doi: 10.1002/acn3.51291

Figure Lengend Snippet: Lymphocyte subpopulations were isolated from fresh PBMC by magnetic separation using specific isolation kits, and leptin receptor expression was measured by RT‐PCR (A) and flow cytometry (B). Cells were cultured (5 × 10 4 cells/well) in round bottom 96‐well plates for 72 h and stimulated as follows: T cells were stimulated with soluble anti‐CD3 and soluble anti‐CD28 (both at 5 µ g/mL concentration); B cells using PMA (5 ng/mL) plus ionomycin (1 μmol/L); and monocytes were activated with 100 ng/mL of LPS. For mRNA expression, data were normalized to the amount of GAPDH, as a control housekeeping gene, using the Pfaffl method. ⁶⁵ Intra‐assay precision was determined in three repeats within one LightCycler run, and interassay variation was investigated in three different experimental runs. Variations for intertest and intratest experiments were between 5% and 7% in all cases. Flow cytometry data were acquired as described in Material and Methods. The results are expressed as Mean Fluorescence Intensity (MFI) of leptin receptor expression (Ob‐Rb; CD295). In all lymphocyte subpopulations, activation significantly increased leptin receptor expression compared to resting cells. Data represent the mean ± SEM from 25 MS patients. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: For blocking experiments human leptin receptor polyclonal antibody (MyBioSource, San Diego CA) was used at a final concentration of 20 μ g/mL; control was an irrelevant isotype‐matched antibody (R&D Systems).

Techniques: Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cell Culture, Concentration Assay, Control, Intra Assay, Fluorescence, Activation Assay

Journal: Annals of Clinical and Translational Neurology

Article Title: Obesity and the risk of Multiple Sclerosis. The role of Leptin

doi: 10.1002/acn3.51291

Figure Lengend Snippet: (A) Concentration curve of antiapoptotic effect mediated by leptin. MBP 83–102 peptide‐specific T cells were cultured in serum‐free culture medium for 24 h, in the presence and absence of different concentrations of leptin. Maximal antiapoptotic effects were seen at 250 ng/mL. Inhibition of apoptosis was leptin dependent, since leptin receptor silencing using siRNA abrogated the leptin effect. Jurkat T cells were used as positive control, with the maximum inhibition of apoptosis reached at concentrations significantly lower than those necessary to prevent apoptosis in autoreactive T cells. Data represent mean values ± SEM of triplicate cultures from five independent experiments. (B) Leptin decreases apoptosis induction in MBP 83–102 , peptide‐specific T cells from MS patients. Three days after Ag stimulation, autoreactive T cells were cultured for 24 h in serum‐free medium, with and without leptin (250 ng/mL). Both antileptin receptor and control antibodies were added at a final concentration of 20 µ g/mL, 30 min before leptin (250 ng/mL). The antiapoptotic effect of leptin was blocked by antileptin receptor mAb, but not modified by an isotype control antibody. MBP 83–102 peptide‐specific T cells in which leptin receptor was silenced using siRNA technique were included in this assay as a negative control. (C) Leptin at a concentration of 50 ng/mL decreases apoptosis induction in Jurkat T cells, cultured in conditions similar to MBP 83–102 peptide‐specific T cells. Data represent mean ± SEM from seven different experiments performed in triplicate (D) Leptin inhibited steroid‐induced apoptosis in MBP 83–102 peptide‐specific T cells. Three days after Ag stimulation, autoreactive T cells were cultured for 24 h with 10 −6 mol/L hydrocortisone in the presence and in the absence of leptin (250 ng/mL). As in the previous experiment, the antiapoptotic effect of leptin was blocked by antileptin receptor mAb, but not modified by an isotype control antibody. In panels A to D, evidence of apoptosis was evaluated by FITC‐Annexin V and PI staining and analyzed by flow cytometry. (E) Expression of the antiapoptotic molecule Bcl‐2 significantly increased in the presence of leptin. This effect was abrogated in the presence of antileptin receptor mAb, but not modified by an isotype control antibody. (F) Leptin promoted proliferation of both MBP 83–102 , and MOG 63–87 peptide‐specific T cells stimulated with increasing concentrations of the cognate antigen. Cell proliferation was assessed by measuring 3 H thymidine incorporation during the final 12 h of a 60 h culture. As in previous experiments, both antileptin receptor and control antibodies were added at a final concentration of 20 µ g/mL, 30 min before adding leptin (250 ng/mL). Data represent mean ± SEM from five different experiments. For panels B, D, and E each circle represents an individual MBP 83–102 ‐specific T‐cell line, isolated from a total of 15 MS patients. Data represent mean ± SEM. In all experiments, PBMC were isolated from patients with normal BMI, to make sure BMI did not affect cell responsiveness to leptin. Kruskal–Wallis test of one‐way ANOVA and post hoc data analysis applying Dunn's multiple comparison test were performed to analyze differences between groups. LEPR: Leptin receptor; Anti LEPR ab = antileptin receptor antibody **** P < 0.0001.

Article Snippet: For blocking experiments human leptin receptor polyclonal antibody (MyBioSource, San Diego CA) was used at a final concentration of 20 μ g/mL; control was an irrelevant isotype‐matched antibody (R&D Systems).

Techniques: Concentration Assay, Cell Culture, Inhibition, Positive Control, Control, Modification, Negative Control, Staining, Flow Cytometry, Expressing, Isolation, Comparison

Journal: Annals of Clinical and Translational Neurology

Article Title: Obesity and the risk of Multiple Sclerosis. The role of Leptin

doi: 10.1002/acn3.51291

Figure Lengend Snippet: (A) Obese MS patients showed significantly higher numbers of IL‐2, IL‐6, IL‐15, IL‐17, IFN‐γ, and TNF‐α producing cells compared to overweight and normal/underweight MS subjects. Likewise, overweight patients showed a higher number of cytokine‐producing cells compared to normal/underweight MS patients. ** P < 0.01, *** P < 0.001, **** P < 0.0001 (B) MBP 83–102 T‐cell lines isolated from normal weight MS patients, were stimulated with the cognate peptide in the presence of leptin, significantly increased the production of IL‐2, IL‐6, IL‐15, IL‐17, IFN‐γ, and TNF‐α producing cells. These effects were overcome by the addition of an antileptin receptor mAb, but not modified by an isotype control antibody. Stimulation with Ovalbumin 323–339 (20 µ g/mL), as nonrelevant peptide, showed values similar to background. In all experiments, cytokine production was assessed using ELISPOT assays. The specific number of cytokine‐producing cells was calculated by subtracting the numbers of spots obtained in 0 Ag background control cultures, from the number of spots obtained in cultures exposed to stimulating Ag. In both panels, data correspond to the number of spots per 10 5 PBMC from 30 patients, and results represent mean ± SEM. Kruskal–Wallis test of one‐way ANOVA and post hoc data analysis applying Dunn's multiple comparison test were performed to analyze differences between groups. **** P < 0.0001 Anti LEPR ab = antileptin receptor antibody.

Article Snippet: For blocking experiments human leptin receptor polyclonal antibody (MyBioSource, San Diego CA) was used at a final concentration of 20 μ g/mL; control was an irrelevant isotype‐matched antibody (R&D Systems).

Techniques: Isolation, Modification, Control, Enzyme-linked Immunospot, Comparison

Journal: Annals of Clinical and Translational Neurology

Article Title: Obesity and the risk of Multiple Sclerosis. The role of Leptin

doi: 10.1002/acn3.51291

Figure Lengend Snippet: (A) Percentage of CD4 + CD25 + FoxP3 + Treg cells from 90 treatment‐naive RRMS patients were compared to serum leptin levels. Regression analysis showed a statistically inverse correlation between serum leptin levels and the percentage of circulating Treg cells (r = −0.97, P < 0.0001). (B) Fifty thousand CD4 + CD25 + cells isolated from fresh PBMC by magnetic separation using commercially available kits (95% purity, 93% expressing FoxP3) were stimulated with soluble anti‐CD3 and anti‐CD28 (BD Bisociences), both at 5 µ g/mL concentration, in the presence and in the absence of leptin (250 ng/mL). Both antileptin receptor and control isotypes antibodies were added at a final concentration of 20 µ g/mL each, 30 min before adding leptin. Proliferation was determined on day 6 with [ 3 H]‐thymidine added during the final 18 h of culture. Proliferation of CD4 + CD25 + Foxp3 + cells was significantly inhibited after stimulation with leptin (250 ng/mL). This effect was abrogated by the addition of antileptin receptor mAb (LEPR‐ab) but not modified by the control antibody. The addition of exogenous IL‐2 (50 U/mL) reversed Treg‐cell hyporesponsiveness to anti‐CD3/anti‐CD28 stimulation in the presence of leptin. Each circle represents data from an individual patient ( n = 25). Data are presented as mean ± SEM. (C‐E) Inhibitory effects of CD4 + CD25 + FoxP3 + Treg cells were examined in 15 RRMS patients during remission on: proliferative response, and secretion of IFN‐γ, and IL‐17 by Th1 and Th17 polarized MBP 83–102 peptide‐specific T cells. For proliferation assays, CD4 + CD25 + FoxP3 + Treg cells were added together with 2 × 10 4 T‐cell‐depleted irradiated (3000 rad) accessory cells to autologous Th1 or Th17 CD4 + CD25 − MBP 83–102 ‐peptide‐specific effector cells at a ratio 1:1 (10 4 cells/well). Co‐cultures were stimulated with soluble anti‐CD3 (5 µ g/mL) together with soluble anti‐CD28 (5 µ g/mL), in the presence and in the absence of leptin, and proliferation determined in a 60‐hour assay, measuring 3 H‐thymidine incorporation. To measure IFN‐γ and IL‐17 production by CD4 + CD25 − effector cells, supernatants were removed before [ 3 H] thymidine addition, and analyzed using commercially available ELISA kits. The addition of leptin to the cultures (250 ng/mL) abrogated the inhibitory effects mediated by CD4 + CD25 + FoxP3 + Treg cells. For panels C, D, and E, each circle represents values for an individual T‐cell line (mean of triplicate cultures). Data represent mean ± SEM. Kruskal–Wallis test of one‐way ANOVA and post hoc data analysis applying Dunn's multiple comparison test were performed to analyze differences between groups. Data are presented as mean ± SEM. **** P < 0.0001.

Article Snippet: For blocking experiments human leptin receptor polyclonal antibody (MyBioSource, San Diego CA) was used at a final concentration of 20 μ g/mL; control was an irrelevant isotype‐matched antibody (R&D Systems).

Techniques: Isolation, Expressing, Concentration Assay, Control, Modification, Irradiation, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Annals of Clinical and Translational Neurology

Article Title: Obesity and the risk of Multiple Sclerosis. The role of Leptin

doi: 10.1002/acn3.51291

Figure Lengend Snippet: Mean percentage inhibition of proliferative response, as well as of IFN‐γ and IL‐17 production by CD4 + CD25 + Foxp3 + Treg cells on CD4 + CD25 − MBP 83–102 ‐ and influenza hemagglutinin 307–319 ‐peptide‐specific effector T cells, derived from 15 RRMS during remission was calculated. CD4 + CD25 + FoxP3 + Treg cells were added in variable numbers together with 2 × 10 4 T‐cell‐depleted accessory cells, to a constant number of autologous Th1 or Th17 CD4 + CD25 − effector cells (10 4 cells/well) to achieve appropriate suppressor/responder ratios (1:1, 1:3, and 1:9). Co‐cultures were stimulated with soluble anti‐CD3 (5 µ g/mL) together with soluble anti‐CD28 (5 µ g/mL) in the presence or absence of recombinant human leptin (250 µ g/mL). Proliferation assays and measurement of IFN‐γ and IL‐17 production were performed as described in Figure and in Material and Methods. Proliferative response (A), IFN‐γ production (B) and IL‐17 production (C) were significantly inhibited upon the addition of CD4 + CD25 + FoxP3 + to the CD4 + CD25 − MBP 83–102 effector T cells in a 1:1ratio. Decreasing ratios of suppressor:effector cells (ratios 1:3 and 1:9) resulted in less suppression in all conditions examined. CD4 + CD25 + FoxP3 + exhibited significantly less suppressor activity on CD4 + CD25 − influenza hemagglutinin 307–319 ‐peptide‐specific effector T cells, compare with CD4 + CD25 − MBP 83–102 ‐ peptide‐specific T cells. When leptin was added to cultures, suppression mediated by Treg cells declined significantly, regardless of specific antigen. Assays were performed in triplicate, the symbols represent mean ± SEM. Percentage of CD4 + CD25 + FoxP3 + Treg‐cell inhibition in co‐cultures was defined as: [1‐(Treg:Teff values/Teff values)] × 100. The Mann–Whitney test was used to evaluate differences in CD4 + CD25 + FoxP3 + function between T‐cell lines co‐cultured at different ratios, and differences in suppressor activity exerted by Treg cells on different specific T cells. **** P < 0.0001.

Article Snippet: For blocking experiments human leptin receptor polyclonal antibody (MyBioSource, San Diego CA) was used at a final concentration of 20 μ g/mL; control was an irrelevant isotype‐matched antibody (R&D Systems).

Techniques: Inhibition, Derivative Assay, Recombinant, Activity Assay, MANN-WHITNEY, Cell Culture

Journal: Annals of Clinical and Translational Neurology

Article Title: Obesity and the risk of Multiple Sclerosis. The role of Leptin

doi: 10.1002/acn3.51291

Figure Lengend Snippet: In panels A to F cells were stimulated with soluble anti‐CD3/anti‐CD28 (5 µ g/mL each) during 6 h, in the presence and in the absence of leptin (A) Stimulation of CD4 + CD25 − MBP 83–102 effector T cells in the presence of leptin significantly increased p‐STAT3 Y705 levels. (B) In contrast, no differences were observed in CD4 + CD25 + FoxP3 + Treg cells under similar experimental conditions. (C) Stimulation of CD4 + CD25 − MBP 83–102 effector T cells in the presence of leptin, induced a significant increase in p‐ ERK1 T202/Y204 /ERK2T 185/Y187 expression. (D) Conversely, Treg cells stimulated under similar conditions showed a marked decrease in p‐ ERK1 T202/Y204 /ERK2T 185/Y187 . (E‐F) Using similar experimental conditions for both CD4 + CD25 − effector cells and for CD4 + CD25 + FoxP3 + Treg cells, a marked decrease in expression of cell cycle inhibitor p27 kip1 was observed in the former, whereas p27 kip1 was significantly increased in Treg cells. Leptin‐mediated effects were abrogated by antileptin receptor antibody, but not modified by an isotype control (20 µ g/mL). Each circle represents an individual MBP 83–102 ‐specific T‐cell line isolated from a total of 15 MS patients. Data represent mean ± SEM. Kruskal–Wallis test of one‐way ANOVA and post hoc data analysis applying Dunn's multiple comparison test were performed to analyze differences between groups. Anti LEPR ab = antileptin receptor antibody. **** P < 0.0001.

Article Snippet: For blocking experiments human leptin receptor polyclonal antibody (MyBioSource, San Diego CA) was used at a final concentration of 20 μ g/mL; control was an irrelevant isotype‐matched antibody (R&D Systems).

Techniques: Expressing, Modification, Control, Isolation, Comparison

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/eLife.38089

Figure Lengend Snippet: ( a ) Schematic representation of erythropoietin receptor (EPOR) and leptin receptor (LEPR) bound to JAK2. Each receptor binds to JAK2 via a box1 interaction with the FERM domain, and a box2 interaction with the SH2 domain. ( b,c ) The crystal structures of EPOR and LEPR bound to JAK2 at 2.65 and 2.83 Å respectively. ( b ) Cartoon representation of residues 279 to 335 of EPOR bound to JAK2 FERM–SH2. JAK2 is shown in blue, and EPOR shown in orange. ( c ) Cartoon representation of residues 866 to 885 of LEPR bound to JAK2. JAK2 is shown in blue, and LEPR shown in yellow.

Article Snippet: Antibody ( Capra aegagrus ) , Anti-human leptin receptor polyclonal antibody , R and D systems , AF497 , Concentration (12.5 ng/uL).

Techniques:

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/eLife.38089

Figure Lengend Snippet: Data collection and refinement statistics.

Article Snippet: Antibody ( Capra aegagrus ) , Anti-human leptin receptor polyclonal antibody , R and D systems , AF497 , Concentration (12.5 ng/uL).

Techniques:

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/eLife.38089

Figure Lengend Snippet: ( a ) Alignment (centered on box1 motif) of intracellular domains of cytokine receptors from the homodimeric/growth hormone and gp130 subfamilies. Sequences start just C-terminal from the transmembrane domain, and end just after the predicted box2 motif. ( b ) Cartoon depiction of the JAK2/LEPR structure, showing the A and B chains (blue) dimerized with symmetry-related chains A’ and B’ (teal). Electron density for LEPR (contoured at 1.0σ) is shown in magenta. ( c ) Cartoon depiction of JAK2/EPOR structure, showing the dimer composed of the A chain (blue) and B chain (teal). In the JAK2/EPOR assymetric unit, there is also a second dimer (C and D chains) that is not shown for clarity. Electron density for EPOR (contoured at 1.0σ) is shown in magenta. ( d ) Alignment of the JAK2/LEPR A-A’ and B-B’ dimers. The A and B chains were aligned to generate the figure. ( e ) Alignment of the JAK2/EPOR A-B and C-D (chains are blue and teal, respectively) dimers. The A and C chains were aligned to generate the figure.

Article Snippet: Antibody ( Capra aegagrus ) , Anti-human leptin receptor polyclonal antibody , R and D systems , AF497 , Concentration (12.5 ng/uL).

Techniques:

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/eLife.38089

Figure Lengend Snippet: ( a ) Alignment of the intracellular receptor sequences of human EPOR and LEPR that interact with JAK2. Sequences begin at the first residue after termination of the transmembrane domain. Sequences were aligned using the φ-Pro-X-Pro motif as an anchor sequence. ( b–e ) Detailed views of interactions between JAK2 and ( b ) EPOR box1, ( c ) LEPR box1, ( d ) EPOR interbox region, and ( e ) EPOR box2. EPOR and LEPR are colored in orange and yellow, respectively, with amino acid side chains shown as sticks. JAK2 is colored blue, with amino acid side chains shown as sticks. Key residues are labeled for reference.

Article Snippet: Antibody ( Capra aegagrus ) , Anti-human leptin receptor polyclonal antibody , R and D systems , AF497 , Concentration (12.5 ng/uL).

Techniques: Residue, Sequencing, Labeling

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/eLife.38089

Figure Lengend Snippet: ( a–c ) Cartoon models comparing the box1-binding sites in the ( a ) apo JAK2 (PDB 4Z32), ( b ) JAK2/EPOR, and ( c ) JAK2/LEPR structures, all shown from the same perspective. The JAK2 cartoon model is shown in dark blue, with key JAK2 residues are shown as sticks in teal. EPOR is shown in orange, and LEPR is shown in yellow. ( d–e ) Cartoon models comparing the ( d ) JAK2/EPOR box1 interaction to the ( e ) JAK1/IFNLR1 box1 interaction (PDB 5IXD). JAK2 is shown in blue with grey transparent surface displayed, with EPOR shown in orange. JAK1 is shown in green with grey transparent surface displayed, and IFNLR1 shown in magenta. Key receptor and JAK residues are labeled and shown as stick models. JAK2 F236 and JAK1 F247 are highlighted.

Article Snippet: Antibody ( Capra aegagrus ) , Anti-human leptin receptor polyclonal antibody , R and D systems , AF497 , Concentration (12.5 ng/uL).

Techniques: Binding Assay, Labeling

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/eLife.38089

Figure Lengend Snippet: ( a ) Top and ( b ) side views of the JAK2/EPOR dimer displayed as a cartoon model, with JAK2 monomers shown in blue and teal, and EPOR shown in orange. Inset box in ( b ) shows a close-up view of the EPOR switch residues Ile282 and Trp283, shown as stick models. Box1 residue Pro287 is also shown for reference. ( c ) Top and ( d ) side views of the JAK2/LEPR dimer displayed as cartoon models, with JAK2 monomers shown in blue and teal as in ( a ) and ( b ), and with LEPR shown in yellow. As in ( b ), the inset box shows a close-in view of the LEPR switch residues 870–872, displayed as stick models. Pro876 from the LEPR box1 sequence is also shown for reference.

Article Snippet: Antibody ( Capra aegagrus ) , Anti-human leptin receptor polyclonal antibody , R and D systems , AF497 , Concentration (12.5 ng/uL).

Techniques: Residue, Sequencing

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/eLife.38089

Figure Lengend Snippet: To highlight the switch/F3 interaction, cartoon depictions of ( a ) EPOR residues Ile262 and Trp263 and ( b ) LEPR residues Leu870, Phe871, and Trp872 interacting with the second JAK2 F3 subdomain are shown. JAK2 is shown in light blue as a cartoon model with stick sidechains, and a grey transparent surface. EPOR and LEPR are shown in orange and yellow, respectively, with the key sidechains shown as stick models. ( c ) A comparable view of homodimeric interaction between Focal Adhesion Kinase FERM F3 residue Trp266 and an opposing F3 subdomain is displayed. The FAK F3 subdomain is shown in purple as a cartoon with stick sidechains displayed, and the interacting F3 loop containing Trp266 is shown as a light pink cartoon, with Trp266 shown as a stick model. ( d ) The bridged dimer interaction seen between chain A and chain B in the JAK2/EPOR assymetric unit are shown. For the JAK2 on the left, shown in light teal, grey transparent surface is also displayed to better highlight the boundaries between the JAK2 molecules. EPOR is shown in orange, with key residues labeled and shown as green stick models. ( e ) A similar interaction interface for JAK2/LEPR chain A and its symmetry related molecule are shown. Only one interface is shown because the second is a symmetry related mirror image. JAK2 is shown as in ( d ), with LEPR shown in yellow with key residues labeled and highlighted in green as stick models.

Article Snippet: Antibody ( Capra aegagrus ) , Anti-human leptin receptor polyclonal antibody , R and D systems , AF497 , Concentration (12.5 ng/uL).

Techniques: Residue, Labeling

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/eLife.38089

Figure Lengend Snippet: Biolayer Interferometry was used to measure equilibrium affinity constants (K D ) for the binding of in vitro translated human LEPR (residues 863–933) containing the listed mutations to wild-type JAK2 FERM–SH2 protein (residues 36–514). The K D ± standard error of three replicate experiments is represented as a bar graph.

Article Snippet: Antibody ( Capra aegagrus ) , Anti-human leptin receptor polyclonal antibody , R and D systems , AF497 , Concentration (12.5 ng/uL).

Techniques: Binding Assay, In Vitro

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/eLife.38089

Figure Lengend Snippet: ( a ) SDS-PAGE gel of in vitro translated receptor peptides. EPOR and LEPR lanes are highlighted in red. ( b–c ) Affinity plots used to calculate steady state affinity (K D ) values for the interaction between JAK2 and biotinylated LEPR peptides (residues 863–933) that contained alanine mutations as the listed positions. Circles and error bars depict the mean response ± SEM for three independent experiments. The ± value associated with each calculated K D is a measure of how well that parameter is defined by the model fitting procedure for that dataset. ( d ) Raw sensorgram data for representative JAK2/LEPR BLI experiments.

Article Snippet: Antibody ( Capra aegagrus ) , Anti-human leptin receptor polyclonal antibody , R and D systems , AF497 , Concentration (12.5 ng/uL).

Techniques: SDS Page, In Vitro

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/eLife.38089

Figure Lengend Snippet: ( a ) Alignment of human and mouse sequences for EPOR and LEPR. Note the numbering differences for mouse versus human (minus one residue for EPOR and minus two residues for LEPR). Residues in the switch region and box1, which are mutated in our experiments, are completely conserved between human and mouse. ( b–c ) Receptor surface staining for stable cell lines expressing ( b ) EPOR variants and ( c ) LEPR variants. Plots represent cumulative events (Y axis) at or below a given staining level (X axis). ( d ) Representative raw flow cytometry histograms showing the phospho-STAT5 (EPOR) or phospho-STAT3 (LEPR) staining in unstimulated cells (black trace) or cells stimulated with cytokine (red trace) as described in Materials and methods.

Article Snippet: Antibody ( Capra aegagrus ) , Anti-human leptin receptor polyclonal antibody , R and D systems , AF497 , Concentration (12.5 ng/uL).

Techniques: Residue, Staining, Stable Transfection, Expressing, Flow Cytometry

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/eLife.38089

Figure Lengend Snippet: ( a,b ) Stable Ba/F3 cell lines expressing wild-type, full length mouse EPOR or LEPR were generated and analyzed for STAT phosphorylation by flow cytometry after stimulation with EPO or leptin. ( a ) Representative plots comparing phospho-STAT5 staining of parental Ba/F3 cells or Ba/F3 cells expressing EPOR. Cells were stimulated with 1 nM mouse EPO for 15 min before fixation, staining, and analysis. ( b ) Representative plots comparing phospho-STAT3 staining of parental Ba/F3 cells or Ba/F3 cells expressing LEPR. Cells were stimulated with 100 pM mouse Leptin for 4 hr prior to fixation, staining, and analysis. ( c ) Analysis of STAT5 phosphorylation by flow cytometry for EPOR wild-type, switch region, and box1 mutants, stimulated with 1 nM EPO as in ( a ). Mean levels of STAT5 phosphorylation were assessed in three separate experiments, with wild-type signal representing 100% in all three experiments. ( d ) Analysis of STAT3 phosphorylation by flow cytometry for LEPR wild-type, switch region, and box1 mutants, stimulated with 100 pM Leptin, as in ( b ). Mean levels of STAT3 phosphorylation was assessed in three separate experiments, with wild-type signal representing 100% in all three experiments. Error bars represent standard error of the mean (SEM).

Article Snippet: Antibody ( Capra aegagrus ) , Anti-human leptin receptor polyclonal antibody , R and D systems , AF497 , Concentration (12.5 ng/uL).

Techniques: Expressing, Generated, Phospho-proteomics, Flow Cytometry, Staining

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/eLife.38089

Figure Lengend Snippet: ( a ) An ‘underside’ view of the JAK2/EPOR (left) and JAK2/LEPR (right) dimers to illustrate the distance between the C-termini (residue 515) of the two JAK2 SH2 domains. Each JAK2 is shown as a cartoon and colored in rainbow from N terminus (blue) to C-terminus (red). EPOR and LEPR are shown as orange and yellow surfaces, respectively. ( b ) ‘Top’ views of JAK2/EPOR (left) and JAK2/LEPR (right) to illustrate the distance between EPOR Trp283 residues or LEPR Trp872 residues in the two dimers. JAK2 monomers are colored in dark and light blue, and EPOR and LEPR are colored in orange and yellow, as above.

Article Snippet: Antibody ( Capra aegagrus ) , Anti-human leptin receptor polyclonal antibody , R and D systems , AF497 , Concentration (12.5 ng/uL).

Techniques: Residue

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/eLife.38089

Figure Lengend Snippet:

Article Snippet: Antibody ( Capra aegagrus ) , Anti-human leptin receptor polyclonal antibody , R and D systems , AF497 , Concentration (12.5 ng/uL).

Techniques: Concentration Assay, Mutagenesis, Produced, Flow Cytometry, Staining, Blocking Assay

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/elife.38089

Figure Lengend Snippet: Figure 1. The structure of the JAK2 FERM–SH2 domain bound to EPOR and LEPR. (a) Schematic representation of erythropoietin receptor (EPOR) and leptin receptor (LEPR) bound to JAK2. Each receptor binds to JAK2 via a box1 interaction with the FERM domain, and a box2 interaction with the SH2 domain. (b,c) The crystal structures of EPOR and LEPR bound to JAK2 at 2.65 and 2.83 A˚ respectively. (b) Cartoon representation of residues 279 to 335 of EPOR bound to JAK2 FERM–SH2. JAK2 is shown in blue, and EPOR shown in orange. (c) Cartoon representation of residues 866 to 885 of LEPR bound to JAK2. JAK2 is shown in blue, and LEPR shown in yellow. DOI: https://doi.org/10.7554/eLife.38089.002 The following figure supplement is available for figure 1:

Article Snippet: DOI: https://doi.org/10.7554/eLife.38089 13 of 21 Continued Reagent type (species) or resource Designation Source or reference Identification Additional information Biological sample (Mus musculus) Leptin R and D systems 498-OB Concentration (100 pM) Antibody (Capra aegagrus) Anti-human leptin receptor polyclonal antibody R and D systems AF497 Concentration (12.5 ng/uL) Antibody (Equus africanus) NorthernLights NL637conjugated anti-goat monoclonal secondary antibody R and D systems NL002 Dilution (1:200) Biological sample (Mus musculus) Epo-Fc fusion Abcam ab170076 Concentration (12.5 ng/uL) Antibody (Mus musculus) Anti-human phospho-Stat3 monoclonal antibody eBioscience/ ThermoFisher 17-9033-41 Dilution (1:40) Antibody (Mus musculus) Anti-human phospho-Stat5 monoclonal antibody eBioscience/ ThermoFisher 25-9010-42 Dilution (1:40) Other Ni-NTA Superflow resin Qiagen 30430 Other Glutathione Sepharose 4B resin GE healthcare 17075605 Other Superdex 200 Hi-load 16/60 column GE healthcare 28989335 Chemical compound, drug EDTA-free protease inhibitors Roche 11836170001 Commercial assay or kit BirA biotinylation kit Avidity BirA500 Commercial assay or kit QuikChange II XL Site-Directed Mutagenesis Kit Agilent 200522 Commercial assay or kit ExiProgen ProXpress PCR Template Kit Bioneer K-7400 Commercial assay or kit ExiProgen EC1 Protein Synthesis Kit Bioneer EK-77161 Chemical compound, drug RPMI-1640 produced in house Chemical compound, drug DMEM produced in house Chemical compound, drug 1X AntibioticAntimycotic Gibco 15240062 Chemical compound, drug 1% NEAA Gibco 11140050 Chemical compound, drug TrypLE Express Gibco 12604013 Chemical compound, drug FugeneHD Promega E2311 Chemical compound, drug Retro-X Concentrator Clontech/Takara 631456 Chemical compound, drug polybrene Millipore TR-1003-G Chemical compound, drug Flow Cytometry Staining Buffer ThermoFisher 00-4222-26 Continued on next page Ferrao et al. eLife 2018;7:e38089.

Techniques:

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/elife.38089

Figure Lengend Snippet: Figure 2. EPOR and LEPR interactions with JAK2. (a) Alignment of the intracellular receptor sequences of human EPOR and LEPR that interact with JAK2. Sequences begin at the first residue after termination of the transmembrane domain. Sequences were aligned using the j-Pro-X-Pro motif as an anchor sequence. (b–e) Detailed views of interactions between JAK2 and (b) EPOR box1, (c) LEPR box1, (d) EPOR interbox region, and (e) EPOR box2. Figure 2 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.38089 13 of 21 Continued Reagent type (species) or resource Designation Source or reference Identification Additional information Biological sample (Mus musculus) Leptin R and D systems 498-OB Concentration (100 pM) Antibody (Capra aegagrus) Anti-human leptin receptor polyclonal antibody R and D systems AF497 Concentration (12.5 ng/uL) Antibody (Equus africanus) NorthernLights NL637conjugated anti-goat monoclonal secondary antibody R and D systems NL002 Dilution (1:200) Biological sample (Mus musculus) Epo-Fc fusion Abcam ab170076 Concentration (12.5 ng/uL) Antibody (Mus musculus) Anti-human phospho-Stat3 monoclonal antibody eBioscience/ ThermoFisher 17-9033-41 Dilution (1:40) Antibody (Mus musculus) Anti-human phospho-Stat5 monoclonal antibody eBioscience/ ThermoFisher 25-9010-42 Dilution (1:40) Other Ni-NTA Superflow resin Qiagen 30430 Other Glutathione Sepharose 4B resin GE healthcare 17075605 Other Superdex 200 Hi-load 16/60 column GE healthcare 28989335 Chemical compound, drug EDTA-free protease inhibitors Roche 11836170001 Commercial assay or kit BirA biotinylation kit Avidity BirA500 Commercial assay or kit QuikChange II XL Site-Directed Mutagenesis Kit Agilent 200522 Commercial assay or kit ExiProgen ProXpress PCR Template Kit Bioneer K-7400 Commercial assay or kit ExiProgen EC1 Protein Synthesis Kit Bioneer EK-77161 Chemical compound, drug RPMI-1640 produced in house Chemical compound, drug DMEM produced in house Chemical compound, drug 1X AntibioticAntimycotic Gibco 15240062 Chemical compound, drug 1% NEAA Gibco 11140050 Chemical compound, drug TrypLE Express Gibco 12604013 Chemical compound, drug FugeneHD Promega E2311 Chemical compound, drug Retro-X Concentrator Clontech/Takara 631456 Chemical compound, drug polybrene Millipore TR-1003-G Chemical compound, drug Flow Cytometry Staining Buffer ThermoFisher 00-4222-26 Continued on next page Ferrao et al. eLife 2018;7:e38089.

Techniques: Residue, Sequencing

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/elife.38089

Figure Lengend Snippet: Figure 3. JAK2/EPOR and JAK2/LEPR dimerization is mediated by the receptor ‘switch’ regions. (a) Top and (b) side views of the JAK2/EPOR dimer displayed as a cartoon model, with JAK2 monomers shown in blue and teal, and EPOR shown in orange. Inset box in (b) shows a close-up view of the EPOR switch residues Ile282 and Trp283, shown as stick models. Box1 residue Pro287 is also shown for reference. (c) Top and (d) side views of the JAK2/LEPR dimer displayed as cartoon models, with JAK2 monomers shown in blue and teal as in (a) and (b), and with LEPR shown in yellow. As in (b), the inset box shows a close-in view of the LEPR switch residues 870–872, displayed as stick models. Pro876 from the LEPR box1 sequence is also shown for reference. DOI: https://doi.org/10.7554/eLife.38089.007 The following figure supplement is available for figure 3:

Article Snippet: DOI: https://doi.org/10.7554/eLife.38089 13 of 21 Continued Reagent type (species) or resource Designation Source or reference Identification Additional information Biological sample (Mus musculus) Leptin R and D systems 498-OB Concentration (100 pM) Antibody (Capra aegagrus) Anti-human leptin receptor polyclonal antibody R and D systems AF497 Concentration (12.5 ng/uL) Antibody (Equus africanus) NorthernLights NL637conjugated anti-goat monoclonal secondary antibody R and D systems NL002 Dilution (1:200) Biological sample (Mus musculus) Epo-Fc fusion Abcam ab170076 Concentration (12.5 ng/uL) Antibody (Mus musculus) Anti-human phospho-Stat3 monoclonal antibody eBioscience/ ThermoFisher 17-9033-41 Dilution (1:40) Antibody (Mus musculus) Anti-human phospho-Stat5 monoclonal antibody eBioscience/ ThermoFisher 25-9010-42 Dilution (1:40) Other Ni-NTA Superflow resin Qiagen 30430 Other Glutathione Sepharose 4B resin GE healthcare 17075605 Other Superdex 200 Hi-load 16/60 column GE healthcare 28989335 Chemical compound, drug EDTA-free protease inhibitors Roche 11836170001 Commercial assay or kit BirA biotinylation kit Avidity BirA500 Commercial assay or kit QuikChange II XL Site-Directed Mutagenesis Kit Agilent 200522 Commercial assay or kit ExiProgen ProXpress PCR Template Kit Bioneer K-7400 Commercial assay or kit ExiProgen EC1 Protein Synthesis Kit Bioneer EK-77161 Chemical compound, drug RPMI-1640 produced in house Chemical compound, drug DMEM produced in house Chemical compound, drug 1X AntibioticAntimycotic Gibco 15240062 Chemical compound, drug 1% NEAA Gibco 11140050 Chemical compound, drug TrypLE Express Gibco 12604013 Chemical compound, drug FugeneHD Promega E2311 Chemical compound, drug Retro-X Concentrator Clontech/Takara 631456 Chemical compound, drug polybrene Millipore TR-1003-G Chemical compound, drug Flow Cytometry Staining Buffer ThermoFisher 00-4222-26 Continued on next page Ferrao et al. eLife 2018;7:e38089.

Techniques: Residue, Sequencing

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/elife.38089

Figure Lengend Snippet: Figure 4. LEPR switch residues are dispensable for binding to JAK2. Biolayer Interferometry was used to measure equilibrium affinity constants (KD) for the binding of in vitro translated human LEPR (residues 863–933) containing the listed mutations to wild-type JAK2 FERM–SH2 protein (residues 36– 514). The KD ± standard error of three replicate experiments is represented as a bar graph. DOI: https://doi.org/10.7554/eLife.38089.009 The following figure supplement is available for figure 4:

Article Snippet: DOI: https://doi.org/10.7554/eLife.38089 13 of 21 Continued Reagent type (species) or resource Designation Source or reference Identification Additional information Biological sample (Mus musculus) Leptin R and D systems 498-OB Concentration (100 pM) Antibody (Capra aegagrus) Anti-human leptin receptor polyclonal antibody R and D systems AF497 Concentration (12.5 ng/uL) Antibody (Equus africanus) NorthernLights NL637conjugated anti-goat monoclonal secondary antibody R and D systems NL002 Dilution (1:200) Biological sample (Mus musculus) Epo-Fc fusion Abcam ab170076 Concentration (12.5 ng/uL) Antibody (Mus musculus) Anti-human phospho-Stat3 monoclonal antibody eBioscience/ ThermoFisher 17-9033-41 Dilution (1:40) Antibody (Mus musculus) Anti-human phospho-Stat5 monoclonal antibody eBioscience/ ThermoFisher 25-9010-42 Dilution (1:40) Other Ni-NTA Superflow resin Qiagen 30430 Other Glutathione Sepharose 4B resin GE healthcare 17075605 Other Superdex 200 Hi-load 16/60 column GE healthcare 28989335 Chemical compound, drug EDTA-free protease inhibitors Roche 11836170001 Commercial assay or kit BirA biotinylation kit Avidity BirA500 Commercial assay or kit QuikChange II XL Site-Directed Mutagenesis Kit Agilent 200522 Commercial assay or kit ExiProgen ProXpress PCR Template Kit Bioneer K-7400 Commercial assay or kit ExiProgen EC1 Protein Synthesis Kit Bioneer EK-77161 Chemical compound, drug RPMI-1640 produced in house Chemical compound, drug DMEM produced in house Chemical compound, drug 1X AntibioticAntimycotic Gibco 15240062 Chemical compound, drug 1% NEAA Gibco 11140050 Chemical compound, drug TrypLE Express Gibco 12604013 Chemical compound, drug FugeneHD Promega E2311 Chemical compound, drug Retro-X Concentrator Clontech/Takara 631456 Chemical compound, drug polybrene Millipore TR-1003-G Chemical compound, drug Flow Cytometry Staining Buffer ThermoFisher 00-4222-26 Continued on next page Ferrao et al. eLife 2018;7:e38089.

Techniques: Binding Assay, In Vitro

Journal: eLife

Article Title: Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

doi: 10.7554/elife.38089

Figure Lengend Snippet: Figure 5. EPOR and LEPR switch residues are required for Epo and Leptin-induced STAT phosphorylation. (a,b) Stable Ba/F3 cell lines expressing wild- type, full length mouse EPOR or LEPR were generated and analyzed for STAT phosphorylation by flow cytometry after stimulation with EPO or leptin. (a) Representative plots comparing phospho-STAT5 staining of parental Ba/F3 cells or Ba/F3 cells expressing EPOR. Cells were stimulated with 1 nM mouse EPO for 15 min before fixation, staining, and analysis. (b) Representative plots comparing phospho-STAT3 staining of parental Ba/F3 cells or Ba/ F3 cells expressing LEPR. Cells were stimulated with 100 pM mouse Leptin for 4 hr prior to fixation, staining, and analysis. (c) Analysis of STAT5 phosphorylation by flow cytometry for EPOR wild-type, switch region, and box1 mutants, stimulated with 1 nM EPO as in (a). Mean levels of STAT5 phosphorylation were assessed in three separate experiments, with wild-type signal representing 100% in all three experiments. (d) Analysis of STAT3 phosphorylation by flow cytometry for LEPR wild-type, switch region, and box1 mutants, stimulated with 100 pM Leptin, as in (b). Mean levels of STAT3 phosphorylation was assessed in three separate experiments, with wild-type signal representing 100% in all three experiments. Error bars represent standard error of the mean (SEM). DOI: https://doi.org/10.7554/eLife.38089.011 The following figure supplement is available for figure 5:

Article Snippet: DOI: https://doi.org/10.7554/eLife.38089 13 of 21 Continued Reagent type (species) or resource Designation Source or reference Identification Additional information Biological sample (Mus musculus) Leptin R and D systems 498-OB Concentration (100 pM) Antibody (Capra aegagrus) Anti-human leptin receptor polyclonal antibody R and D systems AF497 Concentration (12.5 ng/uL) Antibody (Equus africanus) NorthernLights NL637conjugated anti-goat monoclonal secondary antibody R and D systems NL002 Dilution (1:200) Biological sample (Mus musculus) Epo-Fc fusion Abcam ab170076 Concentration (12.5 ng/uL) Antibody (Mus musculus) Anti-human phospho-Stat3 monoclonal antibody eBioscience/ ThermoFisher 17-9033-41 Dilution (1:40) Antibody (Mus musculus) Anti-human phospho-Stat5 monoclonal antibody eBioscience/ ThermoFisher 25-9010-42 Dilution (1:40) Other Ni-NTA Superflow resin Qiagen 30430 Other Glutathione Sepharose 4B resin GE healthcare 17075605 Other Superdex 200 Hi-load 16/60 column GE healthcare 28989335 Chemical compound, drug EDTA-free protease inhibitors Roche 11836170001 Commercial assay or kit BirA biotinylation kit Avidity BirA500 Commercial assay or kit QuikChange II XL Site-Directed Mutagenesis Kit Agilent 200522 Commercial assay or kit ExiProgen ProXpress PCR Template Kit Bioneer K-7400 Commercial assay or kit ExiProgen EC1 Protein Synthesis Kit Bioneer EK-77161 Chemical compound, drug RPMI-1640 produced in house Chemical compound, drug DMEM produced in house Chemical compound, drug 1X AntibioticAntimycotic Gibco 15240062 Chemical compound, drug 1% NEAA Gibco 11140050 Chemical compound, drug TrypLE Express Gibco 12604013 Chemical compound, drug FugeneHD Promega E2311 Chemical compound, drug Retro-X Concentrator Clontech/Takara 631456 Chemical compound, drug polybrene Millipore TR-1003-G Chemical compound, drug Flow Cytometry Staining Buffer ThermoFisher 00-4222-26 Continued on next page Ferrao et al. eLife 2018;7:e38089.

Techniques: Phospho-proteomics, Expressing, Generated, Flow Cytometry, Staining

Journal: International journal of andrology

Article Title: Effect of leptin on motility, capacitation and acrosome reaction of human spermatozoa.

doi: 10.1111/j.1365-2605.2008.00931.x

Figure Lengend Snippet: Figure 1 Immunofluorescent staining of leptin in human ejaculated spermatozoa (Plate a). On the immunopositive cells, leptin immuno- reactivity signal was detected at the equatorial and neck regions. Fluo- rescent photomicrograph of spermatozoa in Fraction I (Plate b) and Fraction P (Plate c) after Percoll separation (·400 magnification) with phase contrast background.

Article Snippet: The smears were then incubated overnight at 4 C in a rabbit polyclonal anti-human leptin antibody (sc-842; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in PBS (1 : 50) and a goat polyclonal anti-human long-form leptin receptor antibody (sc-1832; Santa Cruz Biotechnology) diluted in PBS (1 : 50).

Techniques: Staining

Journal: International journal of andrology

Article Title: Effect of leptin on motility, capacitation and acrosome reaction of human spermatozoa.

doi: 10.1111/j.1365-2605.2008.00931.x

Figure Lengend Snippet: Figure 3 Leptin and leptin receptor mRNA expression in spermato- zoa. In human spermatozoa (S) samples, only signal for GAPDH tran- scripts (housekeeping gene) but not for leptin and the leptin receptors was detected, whereas human placenta (P) samples revealed positive signal for leptin and all the leptin receptor genes in addition to GAPDH.

Article Snippet: The smears were then incubated overnight at 4 C in a rabbit polyclonal anti-human leptin antibody (sc-842; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in PBS (1 : 50) and a goat polyclonal anti-human long-form leptin receptor antibody (sc-1832; Santa Cruz Biotechnology) diluted in PBS (1 : 50).

Techniques: Expressing

Journal: International journal of andrology

Article Title: Effect of leptin on motility, capacitation and acrosome reaction of human spermatozoa.

doi: 10.1111/j.1365-2605.2008.00931.x

Figure Lengend Snippet: Figure 2 Immunofluorescent staining of leptin receptor (long form, ObRL) in human ejaculated spermatozoa. All spermatozoa demon- strated leptin receptor immunoreactivity on the tail region (·400 magnification).

Article Snippet: The smears were then incubated overnight at 4 C in a rabbit polyclonal anti-human leptin antibody (sc-842; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in PBS (1 : 50) and a goat polyclonal anti-human long-form leptin receptor antibody (sc-1832; Santa Cruz Biotechnology) diluted in PBS (1 : 50).

Techniques: Staining

Journal: International journal of andrology

Article Title: Effect of leptin on motility, capacitation and acrosome reaction of human spermatozoa.

doi: 10.1111/j.1365-2605.2008.00931.x

Figure Lengend Snippet: Figure 4 Effect of treatment with graded doses of recombinant human leptin on human sperm capacitation and acrosome reaction (n = 8, no replicate). The percentage of capacitated and acrosome-reacted spermatozoa was determined by the chlortetracycline fluorescent test. All the treatment groups did not show significant difference in the rate of capacitation and acrosome reaction with either 3-h or overnight incubation (Friedman’s test, p > 0.05).

Article Snippet: The smears were then incubated overnight at 4 C in a rabbit polyclonal anti-human leptin antibody (sc-842; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in PBS (1 : 50) and a goat polyclonal anti-human long-form leptin receptor antibody (sc-1832; Santa Cruz Biotechnology) diluted in PBS (1 : 50).

Techniques: Recombinant, Incubation