hnRNP L siRNA knockdown. (A) B16F10 cells were transfected for 24 hours with (i) siRNA control or (ii-v) 5 nM, 10 nM, 20 nM, or 50 nM, respectively, of HNRNP L siRNA. Nuclear protein was extracted and separated by SDS-PAGE (30 μg per lane). The presence of hnRNP L was detected by Western blot with anti-hnRNP L. (B) B16F10 cells were transfected with 50 nM hnRNP L siRNA (left) or control siRNA (right) for 24 hours. The cells were then transfected with 2 μg pcDNA3.1 (–) C6 or C21 exactly as described in panel B. The amount of spliced mRNA (open arrowheads) was calculated using image J³⁸  and expressed as the percentage of total mRNA (nonspliced plus spliced mRNA). (C) The percentage of spliced mRNA obtained following transfection with hnRNP L siRNA or control siRNA. Following treatment with hnRNP L siRNA, the levels obtained for CA21 (○) or CA6 (⋄) are dramatically reduced. After treatment with the siRNA control, the levels obtained for CA21 (•) or CA6 (♦) are not different from that observed in the absence of siRNA. Each data entry represents the mean of triplicate measurements in a single experiment that is representative of 3 independent experiments. (D) Effect of hnRNP L siRNA on B16F10 surface expression of endogenous α2β1. B16F10 cells were transfected twice with 50 nM HNRNP L siRNA (gray curve), 50 nM control siRNA (solid black line), or Lipofectamine 2000 alone (dotted line) over a 72-hour period. Twenty-four hours after the second transfection, flow cytometry was used to quantitate surface α2β1. The results, reported as the GMFI ± SD, are as follows: hnRNP L siRNA, 19.28 ± 0.8; control siRNA, 28.99 ± 1.77; lipofectamine, 31.12 ± 1.46. These results were obtained in 1 representative experiment of 4 independent experiments.