Ab42114

The proteins were extracted from the striatum tissues using radioimmunoprecipitation assay (RIPA) lysis buffer (Ukzybiotech, Beijing, China) according to the manufacturer's instructions. The protein concentration in the lysates was evaluated using a BCA Protein Assay Kit. Proteins were then separated on an SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% BSA-TBST and then incubated with DA1R antibody (ab20066, 1 : 1000), DA2R antibody (ab85367, 1 : 1000), DA3R antibody (ab42114, 1 : 1000), DA4R antibody (ab20424, 1 : 500), DA5R antibody (ab40656, 1 : 200), PKA antibody (ab211265, 1 : 1000), CAM (ab45689, 1 : 1000), and CAMKII (ab52476, 1 : 1000) (Abcam) overnight at 4°C. Then, the blots were washed, incubated with 5% BSA-TBST, and washed again. Consequently, samples were incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. Band detection was performed using the enhanced chemiluminescence (ECL) detection kit. The intensity of the detected bands was calculated densitometrically using the Gel Image system ver.4.00 (Tanon, China).

HEK-293T cells were fixed in 4% paraformaldehyde for 15 min and then washed twice with PBS containing 20 mM glycine before permeabilization with the same buffer containing 0.2% Triton X-100 (5 min incubation). The cells were treated for 1 h with PBS containing 1% bovine serum albumin and labeled with a mouse anti-Rluc (1/100; MAB4400, Millipore) antibody and subsequently treated with Cy3-conjugated anti-mouse (1/200; 715-166-150; Jackson ImmunoResearch (red)) IgG secondary antibody (1 h each). Alternatively, cells were labeled with rabbit anti-D₃R (#ab42114), anti-MT₁R (#ab203038), or anti-MT₂R (#ab115336) antibodies, all from Abcam (Cambridge, UK) and subsequently treated with Cy3-conjugated anti-rabbit (1/200; #711-166-152; Jackson ImmunoResearch (red)) IgG secondary antibody (1 h each). The samples were washed several times and mounted with 30% Mowiol (Calbiochem). Samples were observed under a Leica SP2 confocal microscope (Leica Microsystems).

GBM cells were plated in TIC media at a density of 250,000 cells per dish in T25 tissue culture flasks and cells collected after 24 hours. For immunoblotting, cells were lysed in RIPA buffer (Thermo Scientific 89901) supplemented to 2% SDS and proteins were separated to determine basal expression of Dopamine receptors 2, 3 and 4. Antibodies included those against Dopamine Receptor D2 (AB5084P, Millipore), Dopamine Receptor D3 (ab42114, Abcam), and Dopamine Receptor D4 (pAb-324405, Sigma).