GAPDH used as the internal reference. Each lane contains 30 μ g of total
proteins. The experiment was repeated three times. (B) Statistical data of AKAP95 and Cx43 expression levels
in cells after H89 and Forskolin treatments (20 μ M each). AKAP95 and Cx43 expression levels decreased
in the H89 treatment (p < 0.05) and increased in the Forskolin treatment compared to the control group
(p < 0.05). Images were acquired using the BioRad Chemi DOC XRS + Imaging System, and the gray value
of protein bands was scanned using Image Lab5.0. The ratio of measurements of the treatment group to the
control group was calculated by one-way analysis of variance in SPSS 21.0. (C) Western blot analysis of binding
between AKAP95 and Cx43 in cells after H89 and Forskolin treatments. 500 μ g of extracted total proteins
was co-immunoprecipitated (Co-IP) with 3 μ g of anti-Cx43 antibody. The Co-IP products were resuspended
in an equal volume of loading buffer, and the supernatant was subjected to western blot with anti-Cx43 and
anti-AKAP95 antibodies, and the grayscale value of the bands of the H89 and forskolin groups were compared
with those of the control bands and the ratios were subjected to statistical analysis. (D) Western blot analysis
of binding between AKAP95 and Cx43 after alkaline phosphatase (ALP) treatment. 500 μ g of extracted total
proteins was diluted to 0.2 μ g/μ L with RIPA lysis buffer and split into two groups (500 μ L each). The ALP
treatment was performed following the manufacturer’s instructions (see ALP treatment in M&M). The protein
diluents were subjected to Co-IP with 1 μ g of anti-Cx43 antibody, and the products were resuspended in an
equal volume of loading buffer, and then were subjected to western blot analyzed with anti-Cx43 and anti-
AKAP95 antibodies, the grayscale value of the bands of ALP treatment groups were compared with those of the
control bands and the ratios were subjected to statistical analysis. Results showed that the binding capacity of
Cx43 and AKAP95 was improved after ALP treatment. The experiment was repeated three times.