Table 1.

Effect of boiling and reproducibility of chemiluminescent signals in replicate assays.

Test condition	Cx43	Cx30	Cx26	GAPDH	Actin
a. Boiled/non-boiled ratioa
High glucose cultures	0.43, 0.26	1.41,1.02, 1.27		0.67, 0.66, 0.56
Low glucose cultures	0.35, 0.13	1.13, 0.94, 0.88	0.81, 0.74, 0.32	0.60, 0.43, 0.36	0.30, 0.34
b. Duplicates on same gelb	1.09 ± 0.09 (12, 8%)			1.01, 1.01	1.34, 1.34
c. Replicates on different gelsc
Pair 1	2.24 ± 0.14 (4, 6%)	1.50, 1.85		1.10 ± 0.15 (5, 14%)	1.43 ± 0.27 (5, 19%)
Pair 2	0.44 ± 0.05 (3, 10%)	0.33, 0.37		0.99 ± 0.24 (4, 24%)	1.17 ± 0.14 (4, 12%)
Pair 3	0.44 ± 0.03 (4, 6%)	0.34, 0.35		0.72 ± 0.28 (5, 39%)	0.53 ± 0.14 (5, 26%)
Pair 4	0.43 ± 0.04 (4, 9%)	0.29, 0.28		0.87 ± 0.15 (5, 18%)	0.59 ± 0.25 (5, 43%)

^aTwo aliquots of an extract from cultured astrocytes were mixed with sample buffer (see Methods), and one sample was boiled prior to loading the same amount of protein from each sample onto different lanes of the same gel. Values are ratios of chemiluminescent signal in the boiled sample to that for the non-boiled sample; each ratio represents a different extract.

^bThe same amount of non-boiled protein was loaded onto two lanes of the same gel, and the relative chemiluminescent signal for each duplicate pair is expressed as a ratio, calculated by dividing the higher value by the lower one. For Cx43, samples from 12 extracts from cultured astrocytes were assayed on different gels; ratios are mean ± SD (n, cv); the coefficient of variation (cv, expressed as percent) = 100*SD/mean. Each ratio for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and actin represents a separate sample.

^cPairs of samples from the inferior colliculus were assayed on the same gel/blot, and the chemiluminescent signal for diabetic sample was normalized to its corresponding control. Ratios for the same diabetic and control sample pair were determined in 3–5 replicate blots from either (i) pair-wise assays of one diabetic and one control sample on each gel as described in Figs 1 and 2, or (ii) assays for each protein of interest with all diabetic and control samples on the same gel as described in Fig. 4. Ratios are either the mean ± SD (n, cv) for the number of samples for diabetic-control sample pairs or values for separate samples. Note that, on average, the cv for replicate diabetic/control ratios was higher for actin and GAPDH than for Cx43 and for the duplicate assays of Cx30. Note that pair 1 represents the diabetic outlier sample from the one rat that had high levels of Cx43 and Cx30 compared to the other three diabetic rats that had Cx43 and Cx30 levels lower than those of the controls (see Fig. 4).