Mutant KIT Constructs and Transient Transfection
MUTANTS
Based on the KIT wild-type (WT) eukaryotic expression vectors, 4 KIT mutants were established with the QuikChange Lightning Site-Directed Mutagenesis KIT (#210518), including V559D + V654A, V559D + D820A, V559D + N822K, A502_Y503dup + N822K. All mutants were confirmed by Sanger sequencing. HEK293T cells were transiently transfected with the above 4 mutants and were treated with anlotinib for 4 h after 40-48 h of normal culture. Protein lysates were prepared for western blotting to test the biochemical activity of anlotinib at inhibiting the KIT kinase.