Fig. 2

CAMKK2 is a direct interacting protein of bufalin and highly expressed in ICC. A Representative proteome microarrays results. Bufalin–protein interaction was detected (bule boxes) between bufalin and CAMKK2 by red fluorescence. Representative images of protein microarray showed positive control (blue arrow), negative control (yellow arrow) and CAMKK2 spot (red arrow) on the enlarged images. B Molecular docking predicted the potential binding sites of bufalin and CAMKK2. C Western blot analysis of CAMKK2 expression in HCCC-9810, RBE, and QBC-939 cells treated with different concentrations bufalin. D The quantification of western blot. Protein levels were normalized to tubulin. E Bufalin is labeled with biotin, pull down and verify that CAMKK2 is the target of bufalin. F CAMKK2 has stronger binding force than ATP1A1. G Pan-cancer analysis CAMMK2 was highly expressed in ICC, with red representing tumors and blue representing normal tissues. H Western blot analysis of CAMKK2 expression in HCCC-9810, RBE, L02 and QBC-939 cells. I The quantification of western blot (H). J The expression and cell localization of CAMKK2 in HCCC-9810, RBE, L02 and QBC-939 cells were analyzed by IF. K The expression level of CAMKK2 in tumor tissues and normal tissues. L mRNA expression levels of CAMKK2 in tumor tissue and normal tissue. M In the TCGA database, the top 6 signaling pathways related to CAMKK2 were enriched. All results were presented as the mean ± SD (n = 3). *p < 0.05, **p < 0.01