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Fig. 6. Depletion of ABBA by siRNA results in glial process extension defects. (A) Time-lapse images from C6-R cells expressing GFP-tagged ABBA IM domain and GFP-tagged full-length protein. Cells expressing the IM domain typically displayed multiple protruding microspikes (black arrowhead), whereas the full-length protein localised to the plasma membrane but did not induce dramatic microspike formation. (B) Western blot analysis demonstrating ABBA protein levels before and after transfection with siRNA oligonucleotides. Anti-actin antibody was used as a loading control. (C) Representative images derived from CellIQ cell imaging platform at 2 and 12 hour time points after replating control siRNA oligonucleotide duplex or with siRNA specific to ABBA-transfected C6-R cells. Scale bar: 5 µm. (D) Graph of mean lengths of C6-R cell processes from six individual wells per group. More than 50 cells were counted in each well. Error bars represent s.e.m. (E) Western blot analysis demonstrating the resistance of ABBA-GFP rescue construct to ABBA siRNA. (F) Quantification of cell lengths from control siRNA or ABBA siRNA-transfected cells that were cotransfected with GFP or siRNA-resistant GFP-ABBA constructs 1.5 hours after replating. Data are represented as mean ± s.e.m. (n=49). Statistical significance was established by one-way ANOVA using Tukey test for comparison.
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