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. 2010 Sep 23;5(9):e12908. doi: 10.1371/journal.pone.0012908

Figure 6. Tubulin deacetylase inhibition disrupts the axon initial segment specific enrichment in detergent resistant acetylated microtubules.

Figure 6

(A) Acetylated-α-tubulin levels in microtubules resistant to PHEM buffer extraction in control and TSA treated neurons. (B) Graph represents the mean ± SEM of α-tubulin acetylation levels from 3 experiments as indicated in A. **p<0.01, *** p<0.001, t-test. (C) Acetylated-α-tubulin expression in 7 DIV hippocampal neurons. Axon initial segment is stained with the pIκBα antibody. (D) 7 DIV hippocampal neurons fixed after extraction with 0.5% Triton X-100 in cytoskeletal buffer (2 mM MgCl2, 10 mM EGTA, 60 mM Pipes pH 7.0) for 5 min at 37°C. Arrows indicate the axon initial segments, stained with acetylated-α-tubulin (red) and pIκBα (green). Box shows an amplification of the indicated axon initial segment. (E) Acetylated-α-tubulin localization in 100 nM TSA treated neurons extracted as indicated in D. Note that acetylated tubulin is not further restricted to the AIS and located along the axon. (F) Acetylated-α-tubulin and total α-tubulin immunostaining of control or 100 nM TSA treated neurons extracted with 0.5% Triton X-100. Arrows indicate the position of neuronal somas. (G) Graph represents the fluorescence intensity of acetylated-α-tubulin (red lines) and α-tubulin (green lines) along the stained axon in control (dotted lines) and TSA (straight lines) treated neurons shown in F. Intensity lines are the result of smoothing the data obtained with the ImageJ program using the Sigmaplot software.