Figure 3.

Requirement of TH phosphorylation at Ser-31 for interaction with vesicles. A, relative amount of total TH and THSer(P)-31 in PC12* microsomal fractionation analyzed by Western blotting after inhibition of Ser-31 phosphorylation with 50 μm roscovitine and 50 μm SL327 (R/S). Bars, average relative amount of TH or THSer(P)-31 compared with the untreated samples (t test was used for statistical sample comparison; ***, p < 0.001; data are represented as mean ± S.D. (error bars); n = 3). Representative blots are shown. B, relative amounts of markers to determine the vesicle biogenesis status in microsomal fraction treated or not with R/S. The markers analyzed were GM130 for GC, clathrin, and transferrin receptor (TfR) for endosomes; VMAT2 for dopamine-vesicles; and SPC25 (signal peptidase complex 25) for endoplasmic reticulum. Equal amounts of treated and untreated samples were loaded, as shown in the total protein loading (right). C, relative amount of recombinant V5-TH1-WT, V5-TH1-S31A, or V5-TH1-S31E overexpressed in microsomal fractions of neuroblastoma cells. Microsomal fractions were analyzed by Western blotting using the V5 tag for detection, and mutant THs were compared with WT (representative blots are shown). Bars, relative amount of the WT construct compared with the mutant proteins (t test was used for statistical sample comparison; *, p < 0.05; data are represented as mean ± S.D.; n = 3). D, relative amount of recombinant V5-TH1-S31E overexpressed in microsomal fractions of neuroblastoma cells treated or not with R/S. Microsomal fractions were analyzed by Western blotting using the V5 tag for detection, and treated samples were compared with controls (representative blots are shown). Bars, relative amount of the control samples compared with the treated samples (t test was used for statistical sample comparison, although no significant differences were found; data are represented as mean ± S.D; n = 3).