Figure 9. Signal transduction in tm-chemokine expressing LOX cells upon stimulation with specific antibodies (0.1 µg/ml), but not monovalent F(ab) fragments.
(A) As shown by Western blot after SDS-PAGE, stimulation of tm-CXCL16 or tm-CX3CL1 transfected LOX cells for 20 min with antibodies against the corresponding chemokine domains (0.1 µg/ml), yields a phosphorylation signal for ERK1/2. Non-specific control IgG could not elicit signaling, nor could specific antibodies activate mock-transfected (LOX-pcDNA) cells (positive stimulation control: 2 nM FGF-2, n = 2-3 biological replicates, for corresponding effects in glioma cells compare Figure 9—figure supplement 1). (B) In contrast to the intact specific antibodies, monovalent F(ab) fragments (0.1 µg/ml) obtained by papain digestion and clean-up of these fragments failed to mediate ERK1/2 phosphorylation as demonstrated by Western blot and immunocytochemistry (FGF-2 serves as positive control, n = 3 biological replicates, Bars represent 50 µm).
Figure 9—figure supplement 1. Biological replicates of western blot experiments of stimulations with chemokine-specific antibodies (compare Figure 9).
