Brief, mice were administered MPTP (20 mg/kg in saline, subcutaneously) and probenecid (250 mg/kg in DMSO, intraperitoneally) twice a week for 5 weeks [4]. At the indicated times, brain samples were prepared for histological analyses, RT-PCR/quantitative real time RT-PCR (qRT-PCR) and Western blotting as described. In some experiments, mice were administered tangeretin (10 mg/kg, per oral, in saline including 10 Cremophore EL and 10 DMSO) or the dissolving solution (vehicle) 24 h and 2 h before MPTP/P buy Anlotinib injections [11].RT-PCR and Quantitative Real Time RT-PCR (qRT-PCR)Total RNA was extracted from the ventral midbrain or caudate putamen (CPu) of each mouse using RNAzolHRT (Molecular Research Center Inc, Cincinnati, OH). RT reactions containing 1 mg of total RNA were performed using PrimeScript (Takara, Shiga, Japan). The individual cDNA species were amplified in a reaction mixture containing 1 unit of Taq DNA polymerase (Takara) and specific primers for ATF6, GRP78, ORP150, ATF4, HO-1, CHOP, X-box binding protein 1 (XBP-1), Sec61b, GFAP,Image QuantificationQuantification of the RT-PCR, Western Blotting, and immunohistochemical analyses were performed using Image J (version 1.42, Wayne Rasband, National Institutes of Health). The number of TH positive neurons in the SNpc was counted in five representative sections out of ten sections mounted on one slide, which covered the whole SNpc. Statistical analyses were performed using Bonferroni/Dunn test following a one-way ANOVA.Unfolded Protein Response in Parkinson’s DiseaseFigure 1. The unfolded protein response (UPR) in a mouse model of chronic MPTP/P injection. A, Neurodegeneration (I) and UPR activation (II, III, IV) in the SNpc after MPTP/P injections. Brain sections, including the SN from wild-type mice injected with or without MPTP/P were immunostained with the TH, GRP78, GFAP, and Iba1 antibodies. Scale bars = 50 mm (I), 30 mm (II), 20 mm (III), 20 mm (IV). B, Gene expression in the UPR branches after MPTP/P injections. Total RNA (1 mg) isolated from the ventral midbrain of mice was subjected to RT-PCR with specific primers for ATF6a-target genes (I), ATF4-target genes (II), XBP1-target genes, and b-actin (III). The far right lane in (III) indicates the unspliced and spliced form of the XBP1 from cultured astrocytes treated with thapsigargin (an ER stressor). The relative intensity of the bands MedChemExpress ��-Sitosterol ��-D-glucoside derived from the mice without MPTP/ P injection is designated as one. Values shown are the mean 6 S.D. *P,0.05, **P,0.01 compared with mice without MPTP/P administration (n = 4). doi:10.1371/journal.pone.0047950.gUnfolded Protein Response in Parkinson’s DiseaseFigure 2. Accelerated neurodegeneration and Ub accumulation in ATF6a 2/2 mice after MPTP/P injection. A, TH immunereactivity (I, II) and activated caspase 3 (III) after MPTP/P injections. Brain sections, including the SN (I, III) or CPu (II), from wild-type and ATF6a 2/2 mice that were injected with or without MPTP/P were immunostained with TH and activated caspase 3 antibodies. The number of TH-positive neurons in the SNpc (I) and TH or activated caspase3 intensity in the CPu (II) are shown in the graph. In III, the nuclei are stained with DAPI. Arrows indicate activated caspaseUnfolded Protein Response in Parkinson’s Disease3-positive, TH-positive neurons. The relative number of activated caspase 3-positive, TH-positive neurons in the 
SNpc are also shown in the graph. Values shown are the mean 6 S.D. Scale bars = 50 mm (I), 100 mm (II),.Brief, mice were administered MPTP (20 mg/kg in saline, subcutaneously) and probenecid (250 mg/kg in DMSO, intraperitoneally) twice a week for 5 weeks [4]. At the indicated times, brain samples were prepared for histological analyses, RT-PCR/quantitative real time RT-PCR (qRT-PCR) and Western blotting as described. In some experiments, mice were administered tangeretin (10 mg/kg, per oral, in saline including 10 Cremophore EL and 10 DMSO) or the dissolving solution (vehicle) 24 h and 2 h before MPTP/P injections [11].RT-PCR and Quantitative Real Time RT-PCR (qRT-PCR)Total RNA was extracted from the ventral midbrain or caudate putamen (CPu) of each mouse using RNAzolHRT (Molecular Research Center Inc, Cincinnati, OH). RT reactions containing 1 mg of total RNA were performed using PrimeScript (Takara, Shiga, Japan). The individual cDNA species were amplified in a reaction mixture containing 1 unit of Taq DNA polymerase (Takara) and specific primers for ATF6, GRP78, ORP150, ATF4, HO-1, CHOP, X-box binding protein 1 (XBP-1), Sec61b, GFAP,Image QuantificationQuantification of the RT-PCR, Western Blotting, and immunohistochemical analyses were performed using Image J (version 1.42, Wayne Rasband, National Institutes of Health). The number of TH positive neurons in the SNpc was counted in five representative sections out of ten sections mounted on one slide, which covered the whole SNpc. Statistical analyses were performed using Bonferroni/Dunn test following a one-way ANOVA.Unfolded Protein Response in Parkinson’s DiseaseFigure 1. The unfolded protein response (UPR) in a mouse model of chronic MPTP/P injection. A, Neurodegeneration (I) and UPR activation (II, III, IV) in the SNpc after MPTP/P injections. Brain sections, including the SN from wild-type mice injected with or without MPTP/P were immunostained with the TH, GRP78, GFAP, and Iba1 antibodies. Scale bars = 50 mm (I), 30 mm (II), 20 mm (III), 20 mm (IV). B, Gene expression in the UPR branches after MPTP/P injections. Total RNA (1 mg) isolated from the ventral midbrain of mice was subjected 
to RT-PCR with specific primers for ATF6a-target genes (I), ATF4-target genes (II), XBP1-target genes, and b-actin (III). The far right lane in (III) indicates the unspliced and spliced form of the XBP1 from cultured astrocytes treated with thapsigargin (an ER stressor). The relative intensity of the bands derived from the mice without MPTP/ P injection is designated as one. Values shown are the mean 6 S.D. *P,0.05, **P,0.01 compared with mice without MPTP/P administration (n = 4). doi:10.1371/journal.pone.0047950.gUnfolded Protein Response in Parkinson’s DiseaseFigure 2. Accelerated neurodegeneration and Ub accumulation in ATF6a 2/2 mice after MPTP/P injection. A, TH immunereactivity (I, II) and activated caspase 3 (III) after MPTP/P injections. Brain sections, including the SN (I, III) or CPu (II), from wild-type and ATF6a 2/2 mice that were injected with or without MPTP/P were immunostained with TH and activated caspase 3 antibodies. The number of TH-positive neurons in the SNpc (I) and TH or activated caspase3 intensity in the CPu (II) are shown in the graph. In III, the nuclei are stained with DAPI. Arrows indicate activated caspaseUnfolded Protein Response in Parkinson’s Disease3-positive, TH-positive neurons. The relative number of activated caspase 3-positive, TH-positive neurons in the SNpc are also shown in the graph. Values shown are the mean 6 S.D. Scale bars = 50 mm (I), 100 mm (II),.