Fig. 6: Effect of loss of Shp2 on retinal metabolism.

Immunoblot analysis of 6-week-old Shp2^flox/flox and ^rodShp2^−/− mouse retinas with Glut1, HK-1, HK-2, ALDOC, PKM1, PKM2, p-PKM2, LDHB, LDHA, p-LDHA, PDH, p-PDH, GFAP and actin (A) antibodies. Volcano plot showing differential protein levels of selected proteins from the immunoblots of 6 A, horizontal line indicates changes with (p < 0.05) (B) (n = 4). Quantitative real-time PCR analysis to examine the expression of genes related to inflammation. Equal amounts of retinal mRNA from three independent (n = 3) Shp2^flox/flox and ^rodShp2^−/− mice were used for RT-PCR and normalized by β-actin levels. Volcano plot of differentially expressed inflammatory gene transcripts in ^rodShp2^−/− mice compared to Shp2^flox/flox mice, horizontal line indicates changes with (p < 0.05) (C). ARG1, Arginase 1; CD206, mannose receptor; CD32, cluster of differentiation 32; GFAP, glial fibrillary acidic protein; IL-8, interleukin-8; IL-10, interleukin 10; IL-1β, interleukin-1β; IFNγ, interferon gamma; iNOS, inducible nitric oxide synthase 2; MCP1, monocyte chemoattractant protein-1; NFH, neurofilament heavy chain; TNFα, tumor necrosis factor alpha. LC-MS was used to examine steady-state levels of 126 metabolites in four-independent Shp2^flox/flox and ^rodShp2^−/− mouse retinas that are representative of major pathways in metabolism, including glucose, lactose, amino acids, lipids, and nucleotides. Principal component analysis (PCA) of the samples based on 126 unique compounds demonstrated a clear separation between the Shp2^flox/flox and ^rodShp2^−/− mice (D).