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. 2014 Jun 16;289(33):23141–23153. doi: 10.1074/jbc.M114.562918

FIGURE 5.

FIGURE 5.

Cell proliferation, integrin expression, and affinity for VCAM-1 are not affected by B-Raf knockdown or sorafenib. A, proliferation measured by trypan blue exclusion of Jurkat, shRNA vector control (VC), and B-Raf KD cells cultured with and without doxycycline (±D) for 10 days. B, flow cytometry using a FACSCalibur (BD Bioscience) of isotype control (ISO) (nonspecific mouse IgG) and α4 (mAb L25) or β1 (mAb 33B6) integrin subunits of vector control (VC) and B-Raf KD cells cultured with (+D) and without (NT) doxycycline stained with Alexa Fluor 488 goat anti-mouse secondary antibody. C and D, soluble VCAM-1 (10 μg/ml) binding assay of Jurkat cells incubated for 1 h with sorafenib (50 nm) or methanol vehicle control (C) and vector control (VC) and B-Raf KD cells cultured with doxycycline (+D) or without doxycycline (NT) (D). For C and D, cells were incubated with secondary (2′) AlexaFluor 488 rabbit anti-mouse IgG and no soluble VCAM-1 to show background fluorescence. Error bars, S.E. MFI, mean fluorescence intensity.