hERG1 and VEGF receptors expression and activity in human acute myeloid leukemia cell lines. (A) RT-PCR analysis of herg1 (top panel, 575-bp band), flt1 (second panel, 550-bp band), kdr (third panel, 660-bp band) and gapdh (bottom panel, 138-bp band) transcripts in AML cell lines. Lane ST, molecular weight standard (100 bp; New England Biolabs); lane C+, SH-SY5Y cell line for herg1, HUVEC cell line for flt1 and kdr. (B) hERG1 protein(s) expression on AML cell lines. Cell lysates from AML cell lines, cultured in the presence of serum, were blotted and probed with the anti-pan hERG1 antibody. The top bands, weighing 135 to 150 kDa, refer to the hERG1A isoform; the bottom bands, weighing 75 to 100 kDa, refer to the hERG1B isoform. Reprobing of the membrane with antitubulin antibody is reported in the bottom panel. (C) FLT-1 protein expression in AML cell lines. Cell lysates from cells grown in the presence of serum were blotted and probed with the anti FLT-1 antibody. The arrow indicates the 180 kDa band corresponding to FLT-1. Reprobing of the membrane with antitubulin antibody is reported in the bottom panel. (D) Effect of VEGF₁₆₅ addition on FLT-1 tyrosine phosphorylation. Proteins extracted from AML cell lines, treated with BSA (250μg/mL) or VEGF₁₆₅ (100 ng/mL) for 30 minutes, were immunoprecipitated using anti FLT-1 antibody and the blot revealed using anti p-Tyr antibody. Reprobing the membrane with anti FLT-1 antibody is reported in the bottom panel. Preliminary experiments showed that VEGF₁₆₅ triggered FLT-1 p-Tyr within 15 minutes, reaching a maximum after 30 minutes[b]. IP indicates immunoprecipitation.