Fig. 3

siRNA loss of KLC1 and mutation of LMTK2 WD (1388/1389) disrupt axonal transport of LMTK2. a and b siRNA loss of KLC1 disrupts axonal levels and anterograde axonal transport of LMTK2. Neurons were either untreated (UT) or treated with control or KLC1 siRNAs and then co-transfected with myc-LMTK2 and DsRed to reveal neuronal architecture in (a) or EGFP-LMTK2 alone in (b). a Neurons were fixed and transfected LMTK2 detected by immunostaining for its epitope tag. Representative images of neurons are displayed along with graph showing quantification of relative axonal LMTK2 levels. Scale bar = 20 μm. Data were analysed by Welch’s ANOVA and Games-Howell post hoc test; N = 28–43, error bars are s.e.m., ***p < 0.001. b Representative kymographs showing axonal transport of EGFP-LMTK2 in living neurons; for clarity anterogradely moving EGFP-LMTK2 are illustrated alongside, time = 180 s. Graph shows quantification of anterogradely moving LMTK2 in the different siRNA treated neurons. Data were analysed by Welch’s ANOVA and Games-Howell post hoc test; N = 15–23, error bars are s.e.m., *p < 0.05, **p < 0.01. c and d Mutation of LMTK2 WD (1388/1389) to AA disrupts axonal levels and anterograde axonal transport of LMTK2. c Neurons were co-transfected with DsRed and either myc-LMTK2 or myc-LMTK2(WD1388/1389AA) and LMTK2 detected in fixed cells by immunostaining for its epitope tag. Representative images of neurons are displayed along with graph showing quantification of relative axonal LMTK2 levels. Scale bar = 20 μm. Data were analysed by t-test; N = 28 and 31 neurons, error bars are s.e.m., ***p < 0.001. d Representative kymographs showing axonal transport of EGFP-LMTK2 and EGFP-LMTK2(WD1388/1389AA) in living neurons; for clarity, anterogradely moving EGFP-LMTK2 are illustrated alongside, time = 180 s. Graph shows quantification of anterogradely moving LMTK2 in the different transfected neurons. Data were analysed by t-test; N = 23–26, error bars are s.e.m., *p < 0.05