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. Author manuscript; available in PMC: 2014 Apr 28.
Published in final edited form as: Nat Commun. 2013;4:2690. doi: 10.1038/ncomms3690

Figure 5. Inactivation of GSK3 in the cell body is responsible for augmentation of transcription-dependent axon growth potential downstream of PI3K signalling.

Figure 5

(a) Representative immunoblots of phosphorylated GSK3α-Ser21, GSK3β-Ser9 and CRMP2-Thr514 detected in L4/5 DRG lysates from naïve or injured mice that were subjected to sciatic nerve transection.

(b) Representative immunoblots of phosphorylated GSK3β-Ser9 detected in dissociated adult DRG neurons cultured for either 3 hr or 3 days. For the 3-day cultures, neurons were treated with LY294002 (LY, 10 μM) or vehicle control (DMSO), as indicated.

(c, d) Adult DRG neurons were dissociated, cultured for 3 days, and then replated. Neurons were treated with 6-bromoindirubin-3′-acetoxime (BIO, 500 nM), SB216763 (SB, 10 μM), and/or LY294002 (LY, 10 μM), as indicated, during the initial 3-day-culture period. Replated neurons were cultured in the absence of drugs. Axon length was measured at 20 hr after replating. Representative images of replated neurons are shown in c, and quantification of axon length from three independent experiments is shown in d. Scale bar, 200 μm. Error bars represent s.e.m.** p < 0.01; *** p < 0.001, unpaired two-tailed student t test.

(e, f) Adult DRG neurons were loaded in the somal side of the two-compartment chambers as described in Methods. LY294002 (LY, 10 μM) and/or BIO (500 nM, GSK3i) was locally applied either in the somal (red bars in f) or the axonal side (blue bar in f), as indicated. Y-axis in f depicts the number of microchannels occupied by axons exiting the channels and entering the axonal side (after normalization against the total number of channels). Representative images (e) and quantification (f) of axons entering the axonal compartment are shown. Schematics of the two-compartment chambers (upper left corner) depict where drugs were added. Treatment to the axonal and the somal side is illustrated in blue and red, respectively. Scale bar, 200 μm. n=3, error bars represent s.e.m. * p < 0.01 compared to control; *** p < 0.001; n.s. statistically insignificant difference compared to control, unpaired two-tailed student t test. Original immunoblot images are shown in Supplementary Fig. S11.