Fig. 4

Changes in the phosphorylation of substrates of suggested key kinases and their regulatory proteins by LAT1 inhibition. Protein extracted from cells treated with 30 μM JPH203 and control cells was analyzed by Western blot. (A and B) The decrease in phosphorylation associated with JPH203 treatment for indicated time: Thr-389 of p70 S6K (A) and Ser-1469 of TOP2A (B). The data are presented as a ratio of the signal intensity of phosphorylated protein to total protein ± SD (n = 3). *p value < 0.05. ns = not significant (one sample t test) (C) Decreased phosphorylation of part of CK2 substrates by LAT1 inhibition with JPH203 for 24 h. Phosphorylation on the consensus CK2 substrate motif was detected by a specific antibody. D Decreased phosphorylation of Ser-209 of CK2β, a regulatory subunit of CK2, in cells treated with JPH203 for 24 h. E CK2 activity of BTC cells treated with JPH203. The extracted protein of BTC cells treated with 30 μM JPH203 for 24 h was subject to CK2 activity assay by ELISA using p53 N-terminal peptide and p53-pS46 antibody conjugated with horseradish peroxidase. F The decrease of CK2α co-immunoprecipitated with NOLC1. Protein extracted from KKU-055 and KKU-213 cells treated with JPH203 for 24 h was subject to immunoprecipitation using anti-NOLC1 antibody