Effects of hyaluronidase treatment and blocking of HA-CD44 interaction
on phosphorylation of ERK1/2. A, effects of anti-CD44 treatment
on phospho-ERK1/2. Immunostaining patterns for phospho-ERK1/2 are shown.
Δ3/Δ3, the
Cspg2Δ3/Δ3 fibroblasts. Both WT and the
Cspg2Δ3/Δ3 fibroblasts were cultured in the
presence of an anti-CD44 antibody that blocks HA-CD44 interaction at 1 or 5
μg/ml, as described under “Experimental Procedures.” Note that
treatment with the antibody substantially diminishes the staining intensity
for phospho-ERK1/2 in the Cspg2Δ3/Δ3
fibroblasts (bar, 20 μm). B, effects of hyaluronidase
treatment on phosphorylation of ERK1/2 in WT fibroblasts. The cells were
treated for the periods at the concentrations of hyaluronidase
(HAase) as indicated and applied to immunoblot analysis to detect
phospho-ERK1/2 (pERK), total ERK1/2 (ERK), and actin.
C, phosphorylation levels of ERK1/2. The band density was quantified
using a densitometer. That of phospho-ERK1/2 standardized by that of total
ERK1/2 is shown. D, effects of combined treatment with hyaluronidase
and anti-CD44. The cells were treated with combinations of hyaluronidase and
anti-CD44 (α-CD44) for 2 h at the concentrations indicated and
applied to immunoblot analysis to detect phospho-ERK1/2 (pERK), total
ERK1/2 (ERK), and actin.