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Fig. 3. {alpha}3ß1 is required for MMP-9 mRNA expression. (A) MK+/+ cells (+/+ lane), MK–/– cells (–/– lane) and MK–/– cells stably transfected with the human {alpha}3 subunit (–/–, {alpha}3 lane) or the parental expression vector (–/–, V lane) were cultured on LN-5 ECM for 4 days in serum-containing medium. Total RNA was isolated and assayed by northern blotting with cDNA probes for murine MMP-9 (top panel) or A50 as a control (bottom panel). Two distinct MMP-9 mRNA transcripts of 3.5 kb and 2.7 kb are indicated. (B) RT-PCR was performed using RNA collected as in A as a template. MMP-9 mRNA levels (top panel) and ß-actin mRNA levels (bottom panel) are shown for MK+/+ cells (+/+ lane), MK–/– cells (–/– lane) and MK–/– cells transfected with human {alpha}3 ({alpha}3 lane). (C) FACS analysis with the monoclonal antibody P1B5 confirms high levels of {alpha}3ß1 surface expression in MK–/– cells transfected with the human {alpha}3 integrin subunit (gray peak), but not in untransfected MK–/– cells (white peak).