Physical association between FLT-1 receptor, hERG1, and β₁ integrin subunit in AML cell lines. (A) Co-immunoprecipitation of FLT-1 and hERG1B in AML cell lines. Cell lysates from AML cell lines cultured in the presence of serum were immunoprecipitated with anti FLT-1 antibody; blots were probed with anti-pan hERG1 antibody (top), anti-hERG1B antibody (upper middle), anti-β₁ antibody (lower middle), and anti-FLT-1 antibody (bottom). The pan hERG1 antibody recognizes both hERG1A and hERG1B isoforms.²⁷  Note that with the anti-hERG1B antibody only the higher molecular weight bands are visible, whereas the lower bands can be detected only after long exposure of the membrane (as reported in Lee et al³³ ). Bands relative to the hERG1B protein are indicated by the arrows in the top and middle panels. (B) Co-immunoprecipitation of FLT-1, hERG1B, and β₁ integrin after addition of VEGF, PlGF, and β₁ activating antibody in FLG 29.1 cells. Cells were treated for 30 minutes with BSA (250μg/mL), VEGF₁₆₅ (100 ng/mL), PlGF (50 ng/mL), the β₁ activating antibody TS2/16 (20 μg/mL) or both VEGF₁₆₅ and TS2/16. Proteins were extracted and immunoprecipitated using anti FLT-1 antibody; blot was sequentially revealed using anti-hERG1B antibody (top), anti-pan hERG1 antibody (upper middle), anti-β₁ antibody (lower middle), or with anti FLT-1 antibody (bottom). Inset (left), Densitometric analysis of the amount of hERG1B protein co-immunoprecipitated with FLT-1 after stimulation. The analysis was performed as described in “Materials and methods”; data are the means (± standard error of the mean [SEM]) of 3 separate experiments. *Statistically significant differences between samples are indicated by the horizontal bars. VEGF-treated cells vs BSA-treated cells, Student t test, P = .03; PlGF-treated cells vs BSA-treated cells, Student t test, P = .026; TS2/16-treated cells vs BSA-treated cells, Student t test not significant (NS); VEGF plus TS2/16-treated cells vs BSA-treated cells, Student t test, P = .03; VEGF plus TS2/16-treated cells vs VEGF/PlGF-treated cells, Student t test, NS. Inset (right), densitometric analysis of the amount of β₁ integrin co-immunoprecipitated with FLT-1 after stimulation. VEGF plus TS2/16-treated cells vs TS2/16–treated cells, Student t test, P = .03. (C) Co-immunoprecipitation of FLT-1, hERG1B and β₁ integrin in AML cell lines after addition of VEGF and β₁ activating antibody. AML cell lines were stimulated, and proteins were extracted and immunoprecipitated as noted. Blots were probed with anti pan hERG1 antibody (top), anti-β₁A antibody (middle), and anti-FLT-1 antibody (bottom). Inset, Densitometric analysis of the amount of hERG1B protein co-immunoprecipitated with FLT-1 after stimulation with BSA or VEGF plus TS2/16. The analysis was performed as described in Document S1; data are the means (± SEM) of 3 separate experiments. *Statistically significant differences between samples comprised in the horizontal bars, P < .05, Student t test.