Fig. 5.

a Western blot analysis of total cell lysates using a 5-HT-specific antibody. L6 cells were incubated with 25 µM MDC, 1 µM pargyline (P) or without either of these substances (C), in the absence or presence of 10 µM 5-HT for 1 h. After cell lysis, direct blotting was performed with a 5-HT-specific antibody (N = 3). b SDS gel analysis of total cell lysates after incubation with radioactive 5-HT. L6 cells were incubated with [³H]-labelled 5-HT at 1 and 5 µCi for 1 h, then cells were lysed, and lysates were subjected to SDS gel analysis. Experiments were performed either in the absence or presence of non-labelled 5-HT at a concentration of 10 µM (N = 4). M, radiolabelled molecular weight marker. c Analysis of Rab4 serotonylation using a 5-HT-specific antibody. L6 cells were incubated in the absence or presence of 10 µM 5-HT with or without MDC for 1 h, and lysed in lysis buffer. An aliquot of the lysates was subjected to SDS-PAGE for direct blotting (DB) with an antibody against Rab4, and the remaining lysate was used for immunoprecipitation (IP) with an antibody against Rab4 followed by immunoblotting (IB) with an antibody against beta-actin (ß-Actin) as a loading control and 5-HT respectively. The bar graph shows the quantification of serotonylated Rab4. Bars represent the mean ± S.D. for four independent experiments, with significant differences indicated as ^***P < 0.001, C, Control. d Analysis of Rho serotonylation using a 5-HT-specific antibody. L6 cells were incubated in the absence or presence of 10 µM 5-HT with or without MDC for 1 h, and cells lysed in lysis buffer. An aliquot of the lysates was subjected to SDS-PAGE for direct blotting (DB) with an antibody against Rho, and the remaining lysate was used for immunoprecipitation (IP) with an antibody against Rho followed by immunoblotting (IB) with an antibody against beta-actin (ß-Actin) as a loading control and 5-HT respectively. The bar graph shows the quantification of serotonylated Rho. Bars represent the mean ± S.D. for four independent experiments. C, Control. e Analysis of Rab4 serotonylation using a 5-HT-specific antibody. C2C12 cells were incubated in the absence or presence of 10 µM 5-HT with or without MDC for 1 h, and lysed in lysis buffer. An aliquot of the lysates was subjected to SDS-PAGE for direct blotting (DB) with an antibody against Rab4, and the remaining lysate was used for immunoprecipitation (IP) with an antibody against Rab4 followed by immunoblotting (IB) with an antibody against beta-actin (ß-Actin) as a loading control and 5-HT respectively