Figure 5. Short time effects (2 h) of intravitreal saline, insulin, mitogen-activated protein kinase kinase inhibitor U0126, U0126 plus insulin, phosphatidylinositol 3-kinase inhibitor Ly294002, or Ly294002 plus insulin injections on phospho-Akt/total-Akt and phospho-Erk/total-Erk levels in chicks not wearing any lenses, and after bilateral treatment with +7 D: lenses and −7 D: lenses. The same independent control sample was analyzed on each blot and used for normalization of the data. Band intensities were converted to percentages of the control sample value. The ratio of phospho-form/total-form was then calculated using the normalized values. The bars represent the mean ratio of relative intensity (mean±SEM) between the phospho-forms and non-phosphorylated proteins in percent. Each group included six animals. In control, positive and negative lens–wearing animals (Figure 5A) insulin injections highly increased the retinal ratio of phospho-Akt/total-Akt. In control and animals wearing positive lenses, insulin injections led to an increase in phospho-Erk/total-Erk levels, compared to either the MEK inhibitor or the PI3K inhibitor (Figure 5A). The PI3K inhibitor blocked the stimulatory effect of insulin in control animals without lenses but not in positive and negative lens–treated animals (Figure 5B). The MEK inhibitor significantly blocked the effect of insulin on ERK phosphorylation (Figure 5B). Significant differences were determined with one-way ANOVA and are denoted in the graph (* p<0.05, ** p<0.01, and *** p<0.001).