Figure 4

Characterization of αv integrin expression on microglia derived from wild-type, β3 integrin KO, β5 integrin KO and β3/β5 integrin DKO mice. A. Flow cytometry analysis on microglia derived from wild-type or β3/β5 integrin DKO mice. Microglia were purified from mixed glial cultures as described in Materials and Methods, and then cultured in serum-free medium on vitronectin. After two days in culture, microglial expression of the αv integrin subunit was analyzed by flow cytometry. Note that β3/β5 integrin DKO mice microglia express the αv integrin subunit, though at much reduced levels compared to wild-type cells. B. Biochemical analysis. An αv integrin imunoprecipitation of wild-type microglia revealed a pattern of three bands: αv (140 kD), β5 (90 kD) and β3 (80 kD). As expected, αv imunoprecipitations of β3 KO microglia showed only two dominant bands: αv and β5, while that on β5 KO microglia showed only two dominant bands: αv and β3. Significantly, αv imunoprecipitations of β3/β5 DKO microglia showed that the αv subunit was still present, though at much reduced levels compared to wild-type cells, and in association with weak levels of two β subunits running at the molecular weights of 110 and 80 kD, which correspond to β1 and β8 integrin subunits, respectively. C. Confirmation that microglia express the αvβ8 integrin. Immunoprecipitations of DKO microglia with a β8 integrin polyclonal antibody detected a pattern of two bands running at 140 kD and 80 KD, that co-migrate with the αv and lower β integrin subunit detected in the αv immunoprecipitation. This confirms that the extra 80 kD band expressed by microglia is the β8 integrin subunit.