Evaluation of anti-GHS-R Mab specificity.

<p>A. Western blot of analysis of a selected brain region (hypothalamus) and a non-brain GHS-R bearing tissue (heart) showing a single band of the predicted molecular weight for GHSR at 48 kDa B. Immunoprecipitation of rat cortical (C) and hippocampal (H) primary neurons lysates with Mab 1D8B2 and commercial polyclonal antibody. Lysates from rat hippocampal and cortical neurons after nine days in vitro were immunoprecipitated with Mab (8 µg) or polyclonal antibody (CTRL), resolved and transferred to nitrocellulose membranes. The western blot analysis were performed with the polyclonal antibody when the monoclonal was used for immunoprecipitation and the other way around, both immunoprecipitation analysis clearly showed a 48 kD band corresponding to the predicted size of GHS-R. β-actin was used as a loading control. Sizes (kD) of molecular mass markers are indicated on the right.</p

Immunofluorescence staining on primary neuronal cultures.

<p>Immunofluorescence staining with GHS-R of either cortical (CN) or hippocampal (HN) neurons at different stages of development. Both neuronal cultures show correct maturation in vitro, as assayed by positive staining of vesicular marker for excitatory neurotransmitter transporter Vglut which shows punctuated expression along neuritis at mature stages of development.</p

Comparison of GHS-R levels in cortical and hippocampal neurons.

<p>A. GHS-R mRNA expression levels in cortical (red) and hippocampal (blue) primary neurons normalized on expression levels at 4 div. GHS-R show a significant increase in expression levels at 9 and 16 div, with a significant reduction at later stages (21 div) in both neuronal populations. B. Relative mRNA expression of GHS-R at different developmental stages of hippocampal (red) and cortical (blue) neuronal cells normalized on GAPDH. GHS-R is significantly more expressed in hippocampal rather than cortical neurons at 9 and 16 div.</p

Production and characterization of Mab anti-GHS-R.

<p>A. Immunoprecipitation of 22RV1 cell lysates with Mab 1D8B2 and commercial polyclonal antibody (CTRL). Lysates from cells were immunoprecipitated with Mab (8 µg) or polyclonal antibody, resolved and transferred to nitrocellulose membranes. The western blot analysis were performed with the polyclonal antibody when the monoclonal was used for immunoprecipitation and the other way around, both immunoprecipitation analysis clearly showed a 48 kD band corresponding to the predicted size of GHS-R. Sizes (kD) of molecular mass markers are indicated on the left. These experiments were performed independently at least twice with similar results. B. Binding analysis of Mab 1D8B2 to 22RV1 cells by flow cytometry. Purified monoclonal antibody (6 µg) were analyzed with a flow cytofluorimetric analysis to assess the binding of antibodies to the GHSR on the surface of the cells. The control consisted of an anti-GHSR purified polyclonal antibody. Experiments were performed four times with reproducible results C. Representative image of 22VR1 cells cotransfected with GHS-R siRNA and eGFP. eGFP (green) expressing cells do not show positivity for GHSR (red). D. Relative mRNA expression of GHS-R in transfected 22VR1 normalized on GAPDH. GHS-R expression is significantly lower in transfected cells(siRNA GHSR) than in non-transfected cells (GHSR UT).</p