Fig. 3. Growth of cultured astrocytes in high glucose medium differentially affects connexin (Cx) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels.

Western blots of each sample (n=7 independent batches of astrocytes grown in high and low glucose) were assayed in triplicate for each Cx protein by application of different extract protein amounts per gel (2.5, 5, 10, 15, and/or 20 μg/lane, see Figs. 1 and 2). Immunoreactive GAPDH was assayed after stripping each Cx blot and re-probing with antibodies against GAPDH. After background subtraction, the ratio of the signal intensity for each band in the sample derived from astrocytes grown in high glucose to that in cells grown in low glucose was calculated for each protein concentration loaded onto the gel, and the values were averaged for that sample. The mean ratio for each sample pair was used to calculate the overall mean and variance for the seven culture batches grown in high or low glucose for each protein of interest. For Cx26, the signals for the two bands in each sample were combined and used to calculate the ratio for that sample pair. Values for each sample are shown; horizontal lines are mean ratios and vertical bars are ±95% confidence intervals. Statistically significant differences from a ratio of 1.0 (dashed line) were identified by the Wilcoxon signed rank test (one sample t test); P values are indicated.