Fig. 5.

TBCB/TUBA4A interaction and downregulation of TUBA4A protein upon overexpression of TBCB or blocking of endogenous miR-1825. a Anti-myc immunoprecipitation from lysates of HEK293 cells transfected with TBCB-myc or the empty plasmid (pcDNA3) followed by western blotting. Three independent experiments showed similar results. b Densitometric TUBA4A western blot analysis of TBCB-myc and control plasmid (pcDNA3) transfected HEK293 cells (n = 12). Data were normalized to β-actin. A representative western blot is shown in the left panel. c Confocal microscopy images of HEK293 cells expressing TBCB-myc stained for TBCB-myc and TUBA4A using myc- and TUBA4A-specific antibodies, respectively. Mean intensity of TUBA4A immunofluorescence was quantified in TBCB-myc expressing (+) and non-expressing (−) cells on the same slide (n = 14 quantified images from two biological replicates). d Immunostaining of myc and TUBA4A in primary mouse neurons transfected with TBCB-myc and analysis of TUBA4A immunofluorescence intensity in TBCB-myc positive (+) and negative (−) neurons on the same slide (n = 13 quantified images from two biological replicates). e Fluorescence intensity measurement and representative confocal microscopy images of HEK293 cells transfected with antimiR-1825 or the respective control (n = 10 quantified images from two biological replicates) stained for TUBA4A. f Upper panel: representative western blot of HEK293 cells used for quantification shown in the lower panel. Lower panel: densitometric western blot measurement of TUBA4A level relative to β-actin in HEK293 cells transfected with mimic-1825, antimiR-1825 or the respective control RNAs (n = 5; bars in b–f show mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 in a two-tailed Student’s t test; ns not significant, IP immunoprecipitation; scale bars in c–e represent 20 µm)