Fig. 2

Analysis of AMPK phosphorylation, TNF-α expression, and nuclear translocation of NF-κB p65 in D2 and D2G retina and ON. a, b The ratio of pAMPK to AMPK protein in the retina (a), **p = 0.0034, **p = 0.0016; n = 4 per group and ON (b), **p = 0.0018, **p = 0.0013, as analyzed by capillary electrophoresis; n = 6 Keto D2 and 3 D2G ON per group. c Immunohistochemical analysis of TNF-α (green) expression in untreated (Unt) and keto D2 retinas and ONs. Iba1 (red) labels microglia (c) and DAPI (blue) stains nuclei. d, e Percentage of mean fluorescence of TNF-α in the inner retina (d), ***p = 0.0001, and in the ROI of the proximal ON (e), ***p = 0.0001; n = 5 retinae per group, with five sections analyzed per retina. f Western blot analysis of TNF-α in untreated and keto D2 and D2G retinas. g Bar graph showing the quantification by densitometry of TNF-α protein levels normalized to actin levels in the retina, **p = 0.0035, ***p = 0.0001; n = 3 blots per group, each with independent samples. h TNF-α protein levels in ON analyzed by ELISA, **p = 0.0032; n = 8 per group. i Western blot analysis of NF-κB p65 levels in retinal nuclear protein fraction. j Quantification by densitometry of NF-κB p65 levels normalized to total histone H3 levels, **p = 0.0032, ***p = 0.0001; n = 3 blots per group, each with independent samples. k NF-κB p65 levels in nuclear fraction of the ON protein normalized to total histone H3 levels, as quantified with capillary electrophoresis, *p = 0.00593, **p = 0.0023; n = 3 ON per group. l NF-κB p65 (red) immunohistochemistry in the ON. Arrows indicate nuclear translocation of NF-κB. All bar graphs are presented as the mean ± SEM, n = 5–9 analyzed by two-tailed unpaired t test. d, l Scale bar, 20 μm