Samples were homogenized in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% TX, 150 mM NaCl, 2 mM EDTA and protease/phosphatase inhibitor cocktail). Then, 20 µg of homogenate were analysed by western blot (WB) using standard procedures. Membranes were incubated overnight at 4 °C with primary antibodies: TH (1:1000, AB152, Millipore), D1R (1:500, sc-14001, Santa Cruz), D2R (1:500, AB5084P, Millipore), DARPP-32 (1:1000, AB10518, Millipore), P-DARPP-32-Thr34 (1:1000, AB9206, Millipore), LAMP-1 (1:500, sc-19992, Santa Cruz), p62 (1:500, H00008878-M01, Tebu-bio), LC3B (1:1000, NB100-2220, Novus-Bio) and GAD65 (1:1000, BK3988S, Cell Signaling). β-actin (1:5000, MAB1501, Millipore) was used as housekeeping. Immunoreactivity was detected by chemiluminescence and bands quantified by densitometry using ImageJ software. We reported normalized data to the WT group because we calculated the percentage relative to the control for each blot, to correct for any type of differences relative to different gels (for example, recycling of primary antibody or time to ECL exposure). HPLC was performed as previously described64 . Tissue levels of DA (pg/mg wet weight) were used for statistical analysis.
Ab10518
Manufactured by Merck Group
The AB10518 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of this product is to provide a controlled environment for various scientific experiments and procedures. No further details about the intended use or specific applications of this product are available.
Automatically generated - may contain errors
Lab products found in correlation
Ab9206, by Merck Group (2 mentions)
Nb100 2220, by Novus Biologicals (1 mentions)
Mab1501, by Merck Group (1 mentions)
Ab152, by Merck Group (1 mentions)
Ab5084p, by Merck Group (1 mentions)
Sc 14001, by Santa Cruz Biotechnology (1 mentions)
Sc 19992, by Santa Cruz Biotechnology (1 mentions)
Chemiluminescence detection reagent, by PerkinElmer (1 mentions)
β tubulin, by Merck Group (1 mentions)
2 protocols using ab10518
1
Western Blot Analysis of Dopaminergic Markers
2
Western Blot Analysis of Dopamine Signaling
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Muscle and NAc samples were pulverized and homogenized as described previously [39] (link), and protein concentrations were determined via the Bradford protein assay. Proteins (50 μg) were resolved by SDS-PAGE for Western blot analysis [40] (link). Antibody-bound proteins were visualized on film using chemiluminescence detection reagents (Perkin–Elmer Life Sciences). Protein bands were scanned, and quantitated by densitometry (ImageJ, National Institute of Health). Commercially available primary antibodies were against DARPP32 (AB-10518); pThr34-DARPP32 (AB-9206); D1R (ABN20; all Millipore, Temecula, CA); TH (2792), pThr75-DARPP32 (2301), ΔFosB (9890; all Cell Signaling Technology, Danvers, MA); D2-type dopamine receptor (D2R; ab85367; Abcam, Cambridge, MA); and glucose transporter 4 (GLUT4; sc-7938), hexokinase 2 (HXK2; sc-6521; all Santa Cruz Biotechnology, Dallas, TX). Loading was controlled using glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25337; Santa Cruz Biotechnology) or β-tubulin (05–661; Millipore, Temecula, CA).
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