Figure 1.

Distribution of TH phosphorylated forms. A–C, HEK293 cells were transfected with the WT or the phospho-null V5-TH1-S19A, V5-TH1-S31A, and V5-TH1-S40A or the phospho-mimicking V5-TH1-S19E, V5-TH1-S31E, and V5-TH1-S40E constructs. Phosphorylation at Ser-19, Ser-31, or Ser-40 was detected by Western blotting, loading equal amounts of total protein. Total TH (THt), V5 (transfection control), and GAPDH (loading control) detection was also carried out. D, immunofluorescence (top panels) of THSer(P)-31 in PC12* cells treated or not with R/S to inhibit Ser-31 phosphorylation and corresponding Western blot analysis (bottom panel), where total TH and GAPDH were also detected as controls. Arrows, perinuclear signal. E, immunofluorescence of total TH, THSer(P)-19, and THSer(P)-40 in PC12* cells treated or not with R/S. F, cellular distribution of THSer(P)-31 in PC12Adh. Arrows, perinuclear signal. G, cellular distribution of THSer(P)-31 in human pluripotent induced dopaminergic neurons (iCell DopaNeurons). Arrows, perinuclear signal. H, THSer(P)-31 distribution in PC12* stimulated with 50 ng/ml 2.5S NGF for 48 h. Insets, enlarged numbered areas. In all images, 10-μm scale bars are shown, and all nuclei are stained with DAPI (blue). I, whole-lysate Western blot of PC12* and PC12Adh cell lines stimulated or not with NGF and detection of dopamine-related marker proteins such as DOPA β-hydroxylase (DBH), chromogranin A (ChrA), total TH, THSer(P)-31, and vesicular monoamine transporter 2 (VMAT2). Equal amounts of protein were loaded as shown in the total protein loading represented in the right panel.