Figure 3

Serine 383 and Serine 389 are critical to Elk-1’s function in axon growth. (a) Expression of Flag in HEK 293T cells at 3 days after transfection with WT and double mutants Elk-1 OE plasmids. This WB shows equivalent expression levels of the mutant plasmids. Full blot in Supplemental Fig. S2. (b) Effect of the double mutant Elk-1 OE plasmid on Elk-1 localization in vitro. Nuclear-cytoplasmic fractionation was performed 3 days after transfection of HEK 293T cells with Flag-tagged plasmids. Western blots of Flag of cytoplasmic and nuclear extracts were performed. (c, d) Quantitative analysis of (b). Flag intensity indicates localization of Elk-1 to cytoplasm and nucleus. n = 4, *P < 0.05, **P < 0.01, Kruskal–Wallis test, followed by two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for multiple testing. (e) Representative images show neurite length of hippocampal neurons 3 days after transfection with WT and double mutants Elk-1 OE plasmids in vitro. (f) Quantitative analysis of neurite length in (e). n = 33 (WT), n = 23 (S383A/S389A), n = 27 (S383A/S389E), n = 40 (S383E/S389A), n = 25 (S383E/S389E), **P < 0.01, ***P < 0.001, Kruskal–Wallis test, followed by two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for multiple testing. (g) Representative images show RBPMS staining of RGCs at day 14 after optic nerve crush, for either WT AAV2-Elk-1 or S383E/S389A double mutation AAV2-Elk-1 injected WT mice. (h) Quantitative analysis of RBPMS-positive cell number in the retinas in (g). n = 8 per group, two-tailed Student’s t-test. (i) Representative images of the optic nerve sections showing CTB-labelled regenerating axons at day 14 after optic nerve injury, for either WT AAV2-Elk-1 or S383E/S389A double mutation AAV2-Elk-1 injected WT mice. (j) Quantitative analyses of estimated regenerating axons from the injury site in the optic nerves in (i). n = 7 per group, *P < 0.05, multiple t-tests. n.s., not significant. Asterisks, lesion sites. Scale bars, 200 µm. Star, outlier. The data are presented as median ± 95% CI (b, c, e) and means ± S.E.M. (h, j).