Fig. 2: Y136DOPA modification of overexpressed mCh-αSyn detected by a specific antibody.
From: DOPAnization of tyrosine in α-synuclein by tyrosine hydroxylase leads to the formation of oligomers
a A specific antibody for Y136DOPA of αSyn. Y136DOPAmab specifically recognized TH-treated (TH +) but not control (TH-) αSyn, E46K, and A53T in WB analysis. b, c Y136DOPA signals detected in cultured SCG neurons (b) and PC12 cells (c). SCG and PC12 cells overexpressing mCh-αSyn by the lentivirus system were harvested at Day (d) 4 or 8 after infection and continuously examined by WB. The Y136DOPA signal was slightly detected at d4 but significantly increased at d8. βIII-tubulin is a loading control. d Immunofluorescence of PC12 cells overexpressing mCherry (control, upper) or mCh-αSyn (lower) by the lentivirus system. The Y136DOPA signal (green) was detected in cells expressing mCh-αSyn (red) at d8 after infection. Scale bar: 20 µm. e Dopanization of mCh-αSyn in mouse SN neurons. mCh-αSyn was strongly overexpressed in the SNc by the stereotaxic injection of AAV. WB using the SN tissue detected Y136DOPA at 5 weeks after injection. f Immunohistochemistry staining of AAV-injected brain sections. Y136DOPA modification (green) of mCh-αSyn (red) was detected in the cell bodies of TH-positive dopaminergic neurons (blue) in the SNc (upper, white arrowheads), as well as their nerve terminals (white) in the striatum (lower, blue arrows). Scale bars: 20 µm (upper) and 10 µm (lower). g Y136DOPA signals decreased after treatment with the TH inhibitor. PC12 cells expressing mCh-αSyn at d10 after lentiviral infection were incubated with AMPT for 9 h and harvested. WB showed that the Y136DOPA signals were reduced after inhibiting TH enzyme activity. Data are representative of two independent experiments with similar results (a–g).
