Fig. 5.

PS, matricellular receptors, and integrins mediate MB binding and internalization. a Recombinant PS-binding domain from MFG-E8 (PS-BD) was purified and incubated with liposomes with or without PS. Liposomes were then sedimented by centrifugation and levels of bound PS-BD determined by Coomassie staining. b, d, e HeLa cells or purified MBs were pre-incubated with either recombinant GST, PS-BD, or RGD cyclic peptide and its associated negative control RGE. Cells were then incubated with MBs for 3 h, followed by wash and incubation for either 3 h (b and d; to measure MB binding) or 24 h (e, to measure MB internalization). Cells were then fixed and number of MBs counted. Data shown are the means and standard deviations derived from three independent experiments (one-way ANOVA). c HeLa cells were incubated in suspension with RGD or its inactive control RGE. Cells were then plated on fibronectin-coated coverslips. The number of adhered cells were then counted. Data shown are the means and standard deviations derived from three independent experiments (one-way ANOVA). f Purified GFP-MBs were incubated with recombinant purified either GST-PS-BD (left panels) or GST alone (right panels). MBs were then sedimented by centrifugation, washed, fixed, and stained with anti-GST antibodies (one-way ANOVA). g HeLa cells were incubated with recombinant purified GST-PS-BD resuspended in serum-supplemented media. Cells were then washed, fixed and stained with anti-acetylated tubulin antibodies. Arrow in images point to mitotic MB. Asterisk marks extracellular, post-mitotic MB. Boxed region marks the part of the image shown in higher magnification insets below. Scale bar in inset is equivalent to 500 nm