Mer knockdown decreases intracellular survival and proliferation signaling. Wild-type cells were infected with lentiviral particles containing 1 of 2 independent shRNA constructs targeting Mer (shMer1, shMer4) or GFP (shControl) as a nonsilencing control. (A) Mer knockdown was confirmed by immunoblot analysis of whole-cell lysate. Equal amounts of total protein were analyzed to qualitatively demonstrate reduced expression of Mer (∼180 kDa). Blots were stripped and reprobed with anti-actin antibody (∼43 kDa) or anti-tubulin antibody (∼55 kDa) to confirm similar protein loading of 697 and REH lysates, respectively. (B) Wild-type, shControl, and Mer knockdown (shMer1, shMer4) 697 and REH cells were incubated in serum-free medium for 3 hours prior to stimulation with 200 nM Gas6 for 10 minutes or (D) cultured in cRPMI and treated with 30 nM methotrexate (MTX) for 1 hour. Whole-cell lysates were prepared and phosphorylated (denoted by p-) and total proteins were detected. Immunoblots representative of 3 independent experiments are shown. Blots were stripped and reprobed with anti-actin antibody (∼43 kDa) to confirm similar protein loading. (C) Ratio of phosphorylated p38 MAPK and AKT to total protein level was decreased in shMer1 and shMer4 cells relative to shControl cells following stimulation with Gas6. (E) Ratio of phosphorylated ERK1/2 and AKT was decreased in shMer1 and shMer4 cells relative to shControl cells following treatment with MTX.