Journal: Alcohol and alcoholism (Oxford, Oxfordshire)

Article Title: Glycosylation is altered by ethanol in rat hippocampal cultured neurons.

doi: 10.1093/alcalc/agl044

Figure Lengend Snippet: Fig. 4. Western blot analysis of the glucose transporters GLUT1 and GLUT3 in control (CNT) and alcohol-exposed (EtOH) neurons. Histograms show increases in densitometric readings of GLUT1 and GLUT3 in alcohol-exposed cells compared with the controls. The data used for the statistical analysis are the mean ± SD of four independent experiments. Asterisks indicate significant differences (Student’s t-test P < 0.05).

Article Snippet: Antibodies: anti-MAP2 mouse monoclonal antibody (Sigma-Aldrich, Spain); anti-neurofilament (NF) 200 rabbit polyclonal antibody (Sigma-Aldrich); anti-GLUT1 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc. CA, USA), anti-GLUT3 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.), anti-digoxigenin (DIG) mouse monoclonal antibody (Roche Diagnostics, S.L.

Techniques: Western Blot, Control

Journal: The Journal of Neuroscience

Article Title: Structural and Functional Rescue of Chronic Metabolically Stressed Optic Nerves through Respiration

doi: 10.1523/jneurosci.3652-17.2018

Figure Lengend Snippet: Figure 1. Glycogen and glial glucose transporters decreased with glaucoma pathology. 905 A, Glycogen analysis in 3, 6, and 10-month DBA/2J (D2) and control DBA/2J-Gpnmb+ (D2G) optic nerve (ON), 906 (n=6 ON per group). B and F, Capillary electrophoresis of GLUT1 B, and GLUT3 F, protein in 3, 6, and 10-month 907 D2 and D2G ON normalized to total protein then to 3m D2G protein levels (n=8 ON per group). See Figure 1-1. 908 C, Distribution of GLUT1 in ON of 3m D2, and 10m D2G and D2 optic nerves immunolabeled with GFAP 909 (green). Arrows indicate colocalization of GLUT1 and GFAP, (n=3 sections per ON, 6 ON per group). D and E, 910 Glut1 and Glut3 mRNA levels in 3, 6, and 10-month D2 and D2G ON, normalized to Hprt mRNA then to 3m 911 D2G mRNA (n=5-7 ON per group). See Figure 1-1. G, Distribution of GLUT3 in ON stained with Fluoromyelin. 912 Arrows indicate colocalization of GLUT3 with Fluoromyelin, (n=3 sections per ON, 6 ON per group). H and I, 913 Percent of mean fluorescence intensity in the region of interest (ROI) for C, GLUT1 and G, GLUT3. All values are 914 presented as mean ± SEM, one-way ANOVA and Tukey’s post-hoc test. A, F(5, 30) =18.59, **p = 0.0022, ***p 915 = 0.00011; B, F(5, 42) = 6.463, **p = 0.0077; D, F(5, 30) = 1.906, **p = 0.001; E, F(5, 37) = 15.58, **p= 0.0023, 916 ***p =0.0001; H, F(2, 51) = 210.4, ***p = 0.0001. Scale bar=20μm for C and G. 917 918 Figure 2. Monocarboxylate transporters downregulated with glaucoma pathology. 919 A, Mct1 mRNA levels in 3, 6, and 10-month D2 and D2G ON, normalized to Hprt mRNA then to 3m D2G mRNA 920 (n=5-13 ON per group). See Figure 2-1. B, D and E, MCT1, MCT2 and MCT4 protein levels in 3, 6 and 10-month 921 D2G and D2 ON, normalized to total protein and then to 3m D2G protein levels (n=8 ON per group). C, F and G, 922 Distribution of MCT1 C, MCT2 F, and MCT4 G, in 3m D2, and 10m D2G and D2 ON stained with Fluoromyelin 923 (green) or immunolabeled with GFAP (green). Arrows indicate colocalization (n=3 sections per ON, 6 ON per 924 group). H, Distribution of MCT2 in human control and glaucoma patient ON, immunolabeled for β-tubulin 925 (green) and stained with DAPI (blue). Arrows indicate colocalization (n=4 sections per ON, 2 ON per group). 926 See Figure 2-1. I-L, Percent of mean fluorescence intensity in the ROI for I, MCT1, J, MCT2, K, MCT4 D2 and 927 D2G ON, and L MCT2, human ON. M, Quantification of total number of RGC axons in 6m D2, 10m D2G and 928 10m D2 ON. All values are presented as mean ± SEM, one-way ANOVA and Tukey’s post-hoc test. A, F(5, 47) = 929 4.579, *p = 0.0141; B, F(5, 38) = 6.553, **p = 0.0076; D, F(5, 42) = 16.14, *p = 0.0054, **p = 0.0013; E, F(5, 42) 930 = 4.734, *p= 0.0348; I, F(2, 51) = 173.2, ***p = 0.0001; J, F(2, 51) = 402.1, ***p = 0.0001; K, F(2, 51) = 192.6, 931 ***p = 0.0001; L, t(6) = 9.317, ***p = 0.0001, two-tailed unpaired t-test). Scale bar=20μm applies to C, F, G, 932 and H. 933 934 935 Figure 3. Low lactate accompanies AMPK activation and limits mitochondrial biogenesis and metabolic 936 cofactor pools. 937 A, L-lactate levels in 3, 6 and 10-month D2G and D2 ON (n=8 ON per group). B, Ratio of phosphorylated-AMPK 938 (pAMPK) to AMPK protein in 3, 6 and 10-month D2G and D2 ON (n=8 ON per group). C-D, Phosphorylated 939 AMPK immunofluorescence (magenta) and GFAP (green) micrographs in C, human control and glaucoma ON 940 (n=4 sections per ON, 2 ON per group); and D, 3m D2, and 10m D2G and D2 mice (n= 3 sections per ON, 6 ON 941 per group). Arrows indicate colocalization of pAMPK and GFAP. E-F, Percent of mean fluorescence intensity in 942 the region of interest (ROI) for pAMPK in E, 3m D2, and 10m D2G and D2 mice, and F, human. G-I, Analyses of 943 NAD+/NADH, creatine kinase (CK) activity and PGC1-α levels in 3, 6 and 10-month D2G and D2 mice. G, NAD+ 944 normalized to NADH levels (n=6 ON per group). See Figure 3-1. H, Creatine kinase activity normalized to total 945 protein (n=6 ON per group). I, PGC1-α protein levels normalized to total protein levels and then to 3m D2G 946 protein levels (n=8 ON per group). All values are presented as mean ± SEM, one-way ANOVA and Tukey’s post- 947 hoc test. A, F(5, 42) = 22.04, *p = 0.0124; B, F(5, 42) = 35.51, **p = 0.0032, ***p = 0.0001; E, F(2, 45) = 208.4, 948

Article Snippet: List of antibodies used for IHC and Capillary Electrophoresis analyses 1103 Antibody Dilution RRID Company Host AKT1 1:50 for WES AB_329827 Cell Signaling Technology Rabbit AMPK α1 2B7 1:200 for WES AB_2721834 Novus Biologicals Mouse BDNF 1:100 for IHC AB_630940 Santa Cruz Biotechnology Rabbit Brn3a 1:50 for IHC AB_626765 Santa Cruz Biotechnology Mouse βIII-tubulin 1:1000 for IHC AB_107216 Abcam Chicken GLUT1 1:200 for IHC, 1:50 for WES AB_790014 Novus Biologicals Rabbit GLUT3 1:25 for IHC and WES AB_2191306 R&D Systems Mouse Glutamine Synthetase 1:50 for WES AB_1127501 Santa Cruz Biotechnology Mouse GFAP 1:250 for IHC AB_10917109 EMD Millipore Mouse GFAP 1:500 for IHC AB_10013382 Dako-Agilent Rabbit MCT1 1:25 for IHC and WES AB_10841766 Santa Cruz Biotechnology Mouse MCT2 1:50 for WES AB_2721836 Aviva Systems Biology Rabbit 1:50 for WES AB_10855300 Bioss Rabbit MCT4 1:250 for IHC AB_2254765 Santa Cruz Biotechnology Goat 1:50 for WES AB_2189333 Santa Cruz Biotechnology Rabbit NRF2 1:25 for IHC AB_2618882 Developmental Studies Hybridoma Bank Mouse RBPMS 1:200 for IHC AB_10720427 GeneTex Inc. Rabbit PhosphoAKTSer 473 1:50 for WES AB_329825 Cell Signaling Technology Rabbit pAMPK α1Thr 172 1:100 for IHC, 1:50 for WES AB_11032916 Novus Biologicals Rabbit p70S6K 1:50 for WES AB_331676 Cell Signaling Technology Rabbit Phosphop70S6KThr 389 1:50 for WES AB_330944 Cell Signaling Technology Rabbit Phosphop70S6KSer 411 1:25 for WES AB_2182257 Santa Cruz Biotechnology Mouse SOD2 1:100 for IHC AB_2191814 Santa Cruz Biotechnology Mouse TFAM 1:50 for IHC AB_10610743 Santa Cruz Biotechnology Mouse 1104 48 48 1105 Table 2.

Techniques: Control, Electrophoresis, Immunolabeling, Staining, Fluorescence, Two Tailed Test, Activation Assay, Immunofluorescence, Activity Assay