Figure 8

Fractionation and Western blotting analysis of EB1-ΔN2-GFP aggregates. COS-7 cells were transfected with GFP, EB1-GFP or EB1-ΔN2-GFP for 24 h before extraction in detergent-containing buffer and separation into soluble (panels A and B) and insoluble (panels C and D) fractions and analysis by SDS-PAGE and Western blotting. Panel A. GFP immunoblotting of soluble fractions revealed the presence of single immunoreactive bands for GFP, EB1-GFP and EB1-ΔN2-GFP (arrows). Non-specific cross-reacting bands are evident using this polyclonal antibody. EB1 immunoblotting revealed single bands for EB1-GFP, EB1-ΔN2-GFP and endogenous EB1 (arrows). EB3 immunoblotting revealed a single immunoreactive band in all soluble fractions. Panel B. Immunoblotting for p150glued, p50dynamitin and CDIC indicated that the level of these proteins in soluble fractions were similar in all transfections. Panel C. GFP immunoblotting revealed the presence of two immunoreactive bands in the insoluble fraction of GFP transfected cells (arrow and arrowhead), at least three bands in EB1-ΔN2-GFP transfected cells (arrow and arrowheads) and a single weak band for EB1-GFP (arrow). An overexposed image is shown to highlight the accessory bands. EB1 immunoblotting revealed the presence of two immunoreactive bands in EB1-ΔN2-GFP insoluble fractions (arrow and arrowhead). This blot is also shown overexposed. Panel D. Immunoblotting detected p150glued in the insoluble fraction of all extracts (arrow). Two lower molecular weight bands were specifically detected in the insoluble fraction of EB1-ΔN2-GFP transfected cells (arrowheads). The 43 kDa non-specific band present in the GFP and p150glued panels was present in immunoblots performed on insoluble cellular fractions regardless of the primary antibody used.