Fig. 5From: GSK3β-mediated tau hyperphosphorylation triggers diabetic retinal neurodegeneration by disrupting synaptic and mitochondrial functionsReduced tau microtubule binding and microtubule stability are associated with synapse loss in primary RGCs upon glucolipotoxicity in a GSK3β-dependent manner. (a) Representative images of triple immunostaining for RGC-characteristic marker Thy1 (red), neuronal markers TUJ1 (green) and Map2 (blue). Scale bar, 100 μm. Primary RGCs were then exposed to conditioned medium (HG + PA) for 24 h, in the absence or presence of TWS119. (b) Representative images of subcellular expression of pT231-Tau (green) and Thy1 (red) by double immunofluorescence. Scale bar, 20 μm. (c) Representative synaptophysin (green; scale bar, 20 μm) immunostaining in RGCs. Areas boxed in are shown at higher magnification in the lower panels. (d) Western blotting for synaptophysin from whole cell lysates (Total) or synaptosome fractions (Syn). Intensities were quantified and normalized against the level of GAPDH and expressed as percentages of protein abundance under stimulation relative to control. (e) mRNAs of synaptophysin in total lysates or synaptosomes were quantified by Q-PCR. (f) Microtubule sedimentation assay. Western blotting for Tau 5 and β-tubulin in the supernatant (SN) and the microtubule pellet (pellet). Relative intensities of each protein in its respective fraction were quantified and normalized against the sum of the intensity value of that protein (total, including both supernatant and pellet fractions). MT, microtubule. (g) Western blotting for Ac-tubulin in whole cell lysates. Intensities were normalized against the level of GAPDH. (h) Representative images of double immunofluorescence for Ac-tubulin (green) and Tyr-tubulin (red). Scale bar, 40 μm. Data are means ± SEM of three independent experiments. *P < 0.05 and **P < 0.01 vs control; #P < 0.05 and ##P < 0.01 vs HG + PABack to article page