Fig. 4

Co-aggregation analysis of ANT1 with α-synuclein at cellular level. Control/CTRL or 1, untreated SH-SY5Y cells; PD or 2, MPP⁺-treated SH-SY5Y cells. a Morphological effects of MPP⁺ on the viability of neuroblastoma SH-SY5Y cells. Cells were incubated with different concentrations of MPP⁺ ranging from 0 to 4 mmol/L for another 24 h. The morphology of the SH-SY5Ycells were photographed under a bright field optical microscope at the magnification of 200. Scale bar represented 50 μm. b Representative HPLC analysis on cytosolic DA and its standard. c Statistical analysis of cytosolic DA concentration from HPLC analysis between the MPP⁺-treated and untreated SH-SY5Y cells. The statistic analyses were conducted by a two-tailed equal variance Student’s t-test; bars represented the mean  ±  SEM; *P  <  0.05. d Effects of MPP⁺ on the viability of SH-SY5Y cells. Cells were exposed to MPP⁺ with different concentrations ranging from 0 to 8 mmol/L for another 24 h. Cell viability was assessed by MTT assay and expressed as a percentage. e Expression analysis on intracellular TH in SH-SY5Y cells detected by Western blot analysis. Cells lysates containing 60 μg soluble proteins were subjected to Western blot analysis coupled with 12% SDS-PAGE separation. The membrane was probed with monoclonal anti-TH antibody and visualized using a ECL kit. f Densitometric analysis of intracellular TH normalized to β-actin in SH-SY5Y cells. The statistical analyses were performed by a two-tailed equal variance Student’s t test; Bars represented the mean  ±  SEM; *P  <  0.05. g Expression analysis on intracellular ANT1 in SH-SY5Y cells. Eighty microgram of total protein was applied. The membrane was probed with monoclonal anti-ANT1 antibody, and the visualization of bands was performed using ECL. M, Page Ruler prestained protein ladder (Fermentas). h Densitometric analysis of soluble ANT1 normalized to GAPDH in SH-SY5Y cells. The statistical analyses were performed by a two-tailed equal variance Student’s t test; Bars represented the mean  ±  SEM; *P  <  0.05. i Dual immunofluorescence analysis on the co-aggregation of α-synuclein with ANT1 in SH-SY5Y cells. Visualization was performed using an inverted fluorescence microscopy at magnification of 400. Red signal corresponded to the α-synuclein; Green signal corresponded to ANT1; Blue signal corresponded to DAPI-stained nuclei; Co-expression of ANT1 with α-synuclein was shown as the merged images of green and red. The co-aggregation region was indicated by arrows