Figure 1. ApKLC2 and ApKHC1 are induced by 5HT.

1A: Semi-quantitative RTPCR analysis of ApKHC1 and ApKLC2 gene expression. Total RNA was isolated from pleural ganglia or pleural sensory clusters at different time points (0 minutes and 30 minutes) after five pulses of 10 μM 5HT treatments and from untreated controls. ApC/EBP was used as a positive control. ApActin and sensorin mRNAs were used to normalize kinesin mRNA levels; 1B: Fold increase in mRNA levels of ApKHC1 and ApKLC2. The intensity of ApKLC2 and ApKHC1 bands (shown in 1A) were quantified and normalized to sensorin levels using IMAGEQUANT; 1C: Real-Time PCR analysis of changes in transcript levels of ApKLC2 and ApKHC1 in response to 5x 5HT. ApGAPDH mRNA was used for normalization of data. Fold changes were calculated according to Pfaffl, 2001 and 2002; 1D: mRNA in situ analysis of ApKHC1 mRNA expression in sensory neurons (SN) and motor neurons (MN). Confocal projection images are shown; 1E: Quantitation of ApKHC1 mRNA staining in sensory and motor neurons (shown in 1D) by mRNA in situ hybridization. Mean fluorescence intensities of labeled probe hybridizing to targets were quantified by analyzing the confocal images using METAMORPH (n=6, *p<0.01 for both SN and MN, Student’s t test); 1F: Western blot analysis of changes in protein levels of kinesin in response to 5x 5HT. Synaptophysin and β tubulin protein levels were used as loading controls and for normalization of data; 1G: Fold increase in the protein levels of ApKHC and ApKLC (shown in 1F) in 30 minutes of 5HT treatment; 1H: Immunocytochemical analysis of induction of ApKLC and ApKHC protein in 30 minutes of bath application of 5x5HT in sensory neurons. Confocal projection images of two representative examples of immunostaining of ApKHC and ApKLC in sensory neurons are shown; 1I: Quantitation of immunocytochemistry data presented in 2h. Scale bar = 50 μm. Student’s t test was performed to test levels of significance among different populations of specimens. The error bars represent SEM.