Generation of EphA2 Expression Plasmids

An expression plasmid for EphA2 (accession NP_004422.2) was generated by PCR amplification using cDNA of pDONR223-EphA2 (Addgene ID:23926) [24 (link)]. The resulted amplicon was inserted into the pcDNA3 plasmid (Invitrogen). Mutant EphA2 without cytoplasmic domain (EphA2ΔIC) was created by inserting the region of human EphA2 encoding amino acids 1–560 into pcDNA3. EphA2 kinase dead mutant (EphA2K645R-plasmid) was obtained by PCR amplification using primers 5‘-ccggtggccatcaggacgctgaaagcc-3’ and 5‘-ggctttcagcgtcctgatggccaccgg-3’ which replaces Lysine to Arginine. Empty pcDNA3 was used as a control. p85-PI3K expression plasmid was purchased from Addgene (ID:11499) [25 (link)]. Transfection was performed using 1–1.5 μg/ml plasmid per well in 12 well plate using Polyethyleniminde (PEI) and OptiMEM transfection medium (Gibco). Transfection medium was changed after 5 h. Infection was performed at different time points after 20 h of transfection.