Validation of newly defined miR-150 targets. (A) GAB1 or FOXP1 immunoblot analysis in CLL samples of 16 patients selected for low-level (n = 8) vs high-level expression of miR-150 (n = 8). The relative levels of miR-150 expression are indicated (first sample set as 1). The samples were solely chosen based on the differences in miR-150 expression and partially overlapped with samples used in the microarray analysis (7 of 16 samples). The double bands identified by the anti-FOXP1 antibody (JC12 clone) represent 2 splicing variants of FOXP1 as described previously.⁵³  (B) A representative immunoblot for GAB1 or FOXP1 protein in MEC-1 and Raji, primary CLL cells transfected (48 hours) with artificial miR-150 (miR-150, 100 nM), and control miRNA (negative control [Neg. Ctrl], 100 nM). MOCK represents cells treated only with the electroporation pulse. The Blot images were quantified with ImageJ 1.42q (National Institutes of Health), and the GAB1/β-actin and FOXP1/β-actin ratio in MOCK was arbitrarily set at 1. The FOXP1/β-actin rations for its 2 major splicing variants are provided (the ratio of the larger vs the smaller isoform is separated by a slash). (C) The luciferase activity in 293FT cells cotransfected with miR-150 mimic (MIMIC miR-150, 100 nM) or luciferase reporter construct (100 ng) containing the 3′UTR region of GAB1, FOXP1 or GAPDH (GAPDH as control). As a negative control, we used a control miRNA mimic (CTRL Scramble, 100 nM). Luciferase activity was measured 24 hours posttransfection and compared by paired Student t test.