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. Author manuscript; available in PMC: 2023 Sep 1.
Published in final edited form as: Neuroglia. 2023 Jun 21;4(3):158–171. doi: 10.3390/neuroglia4030011

Figure 1. Glucose Transporter-2 (GLUT2) Gene Knockdown Effects on Male and Female Hypothalamic Primary Astrocyte 44/42 kDa MAPK/ERK1/2 Protein Expression and Phosphorylation.

Figure 1.

Hypothalamic primary astrocyte cultures from both sexes were steroid-deprived (18 hr) before pretreatment (72 hr) with scramble (SCR) or GLUT2 short-interfering RNA (siRNA), then incubated (4 hr) in the presence of 5.5 (G5.5) or 0 (G0) mM glucose. Astrocyte lysate aliquots were analyzed in triplicate by Bio-Rad StainFree Western blot equipment and software. Protein optical density (O.D.) measures measured in a Bio-Rad ChemiDoc™ Touch Imaging System were normalized, using Bio-Rad Image Lab™ 6.0.0 software, to total in-lane protein, e.g. all protein electrophoresed in the individual sample lane. Data depict mean normalized total (Figures 1A and 1C) or phosphorylated (Figures 1B and 1D) 44 and 42 kDa ERK1/2 protein O.D. values ± S.E.M. for male (M; gray bars, at left) and female (F; white bars, at right) G5.5- or G0-exposed astrocytes pretreated with SCR versus GLUT2 siRNA. Treatment groups were comprised of male and female astrocytes treated with G5.5/SCR siRNA [solid bars (gray, M; white, F)]; G5.5/GLUT2 siRNA [horizontal-striped bars (gray, M; white, F)]; G0/SCR siRNA [diagonal-striped bars (gray, M; white, F)]; or G0/GLUT2 siRNA [cross-hatched bars (gray, M; white, F)]. Open circles depict individual independent data points. Mean normalized protein O.D. data were analyzed by three-way ANOVA and Student-Newman-Keuls post-hoc test, using GraphPad Prism (Volume 8) software; results are presented in Supplementary Table 1. Statistical differences between discrete pairs of treatment groups are denoted as follows: *p < 0.05; **p < 0.01; ***p < 0.001.