Culturing Murine Fibroblasts and Melanoma Cells

Murine fibroblasts (NR6WT) expressing human epidermal growth factor receptor and modified as previously described^{4 (link)}, were grown in alpha-MEM media supplemented with 1x non-essential amino acids, 1x sodium pyruvate, 1x L-glutamine, 1x pen/strep antibiotics, and 7.5% fetal bovine serum. Parental Melanoma cell line WM1158 and ACTN4 knockdown WM1158 (WM1158 ACTN4 KD)^{20 (link)} cells were grown in DMEM (1 gL⁻¹ glucose):L15 3:1 medium with 10% fetal bovine serum and 1x pen/strip antibiotics. Transfection reagent xFect was purchased from Clontech Life Technologies (Grand Island, NY). Monoclonal antibody against phosphorylated tyrosine (P-Tyr-100) and polyclonal GAPDH antibody were purchased from Cell Signaling Technology (Beverly, MA). Polyclonal GFP antibody was purchased from Abcam (Cambridge MA). Purified bovine muscle actin and polyclonal Actin antibody were purchased from Sigma Aldrich (St. Louis, MO).