Protein interactions between GHSR/DRD1 and GHSR/β-arrestin 2 in mouse brain slices and hippocampal neuron cultures were detected using Duolink Proximity Ligation Assay (PLA) detection kits (Sigma-Aldrich, #DUO92008) following manufacturer’s instructions. The following primary antibodies were used in proper combinations: goat-anti-GHSR (Santa Cruz Biotechnology, #sc-10359, 1 : 100), rabbit-anti-DRD1 (Abcam, #ab81296, 1 : 200), mouse-anti -arrestin 2 (Santa Cruz, #sc-13140). The specificity of antibodies to GHSR and DRD1 was validated as previously described [3 (link)]. The following Duolink in Situ PLA Probes were used: anti-Rabbit PLUS (Sigma-Aldrich, #DUO92002), anti-Goat MINUS (Sigma-Aldrich, #DUO92006), anti-Mouse PLUS (Sigma-Aldrich, #DUO92001). Images were collected on a Nikon confocal microscope. PLA-positive dot number was counted using Nikon-Elements Advanced Research software.
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Quantifying GPCR-Protein Interactions in Brain
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Corresponding Organization : University of Kansas Medical Center
Other organizations : The University of Texas Southwestern Medical Center
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Variable analysis
independent variables
- GHSR/DRD1 protein interactions
- GHSR/β-arrestin 2 protein interactions
- PLA-positive dot number
- Primary antibodies used: goat-anti-GHSR, rabbit-anti-DRD1, mouse-anti-β-arrestin 2
- Duolink In Situ PLA Probes used: anti-Rabbit PLUS, anti-Goat MINUS, anti-Mouse PLUS
- Specificity of antibodies to GHSR and DRD1 was validated as previously described [3]
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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