Fig 2.

Specific expression of integrin αvβ5 in stabilin-2-expressing cells and inhibition of phagocytosis by blocking of the integrin signaling molecule FAK. (A) Surface expression of integrin αvβ3 or αvβ5 on L/Mock, Stab-2/L, or Stab2Δ2 cells. (B) Total protein expression of β5, αv, and β3 in L/Mock and Stab-2/L cells. Total cell lysates were subjected to SDS-PAGE, and expression of integrin subunits was monitored using corresponding antibodies. β-Actin (*) of each cell lysate was assessed as a control. (C) Expression of MFG-E8 and Gas6 monitored by reverse transcription-PCR analysis. Soluble factors MFG-E8 and Gas6 were not expressed in stabilin-2-expressing cells (L/Stab2), whereas Raw264.7 macrophages expressed both MFG-E8 and Gas6. β-Actin expression was measured as a control. (D) After pretreatment with FAK inhibitors, cells were incubated with HEPES-buffered medium (20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl₂, 0.2% BSA) containing aged RBCs plus PF228, PF271, or dimethyl sulfoxide for an additional 1 h at 37°C for phagocytosis assay as described above. NT, nontreated. The phagocytic indexes were determined as the average number of ingested aged RBCs per cell (phagocytic index = number of ingested aged RBCs/total number of cells). The results are expressed as the mean ± SD from at least three experiments. (E) Decreased FAK activity upon pretreatment of FAK inhibitors (PF228 or PF271). Cells were preincubated with medium containing PF228, PF271, or dimethyl sulfoxide (DMSO) over a range of concentrations for 1 h and lysed for immunoblot analysis. Phosphorylated Tyr³⁹⁷ (pTyr³⁹⁷) was detected by anti-FAK pY³⁹⁷ polyclonal antibody and compared with total FAK. (F) Representative images of engulfment of aged RBCs (red arrowheads) in Stab-2/L cells in the presence of FAK inhibitors (PF228 and PF271) and dimethyl sulfoxide as a control.