Figure 1.

Alterations in the level and activity of Lyn result in modulation of DA release in differentiated SH-SY5Y cells. a, ³H-DA release assay. SH-SY5Y cells were preincubated for 30 min with the pan-Src PTKs inhibitor PP2 (50 μm). Cells were depolarized with KCl (100 mm). After 2 min of depolarization, media and cell lysates were separately collected. Data were analyzed as a percentage of DA release and expressed as the mean ± SEM percentage of non-depolarized controls (n = 3). **p < 0.01. b, Infection of SH-SY5Y cells with either the Adv-shLyn or the scrambled sequence control (Adv-SCR). Viruses were used at a concentration of 1 × 10⁷ ifu/ml (predetermined maximal effective concentration, data not shown). Protein lysates were collected after 10 d of infection and Lyn levels were analyzed by Western blot with anti-Lyn antibodies (1:500). Anti-GFP antibodies were used as a method of detecting virus infection, and GAPDH levels were measured to verify equal sample loading (n = 3). c, Time course for knockdown of Lyn protein levels in SH-SY5Y cells infected with either Adv-shLyn or Adv-SCR. As in b, viruses were used at a concentration of 1 × 10⁷ ifu/ml. Lyn levels were analyzed by Western blot as in b. Data are expressed as a percentage of the ratio of Lyn/GAPDH in the control samples (n = 3). ***p < 0.001. d, ³H-DA release assay. SH-SY5Y cells were depolarized with KCl (50 mm, 100 mm). After 2 min of depolarization, media and cell lysates were separately collected. Data were analyzed as the percentage of DA release and expressed as the mean ± SEM percentage of uninfected controls (n = 3). **p < 0.01. Results are expressed as the mean ± SEM percentage of non-depolarized controls (n = 3). **p < 0.01. e, Infection of SH-SY5Y cells with an adenovirus expressing constitutively active Lyn (Adv-CA-Lyn) or control (Adv-GFP). Active Lyn levels were analyzed by Western blot with a pan-active Src PTKs antibody anti-[pY⁴¹⁸]Src. Total Lyn levels were detected using anti-Lyn antibodies; anti-GFP antibodies were used to detect the control virus, and GAPDH levels were measured to verify equal sample loading (n = 3). f, ³H-DA release assay. Cells were infected for 3 d with Adv-CA-Lyn or Adv-GFP, cells were depolarized, and data were analyzed as in d. The results are expressed as the mean ± SEM percentage of uninfected controls (n = 3). ***p < 0.001.