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Fig. 8. MAPK is regulated by ß1 integrin. (A) Western blots show that activation of MAPK by both EGF and FGF2 is reduced by a monoclonal anti-ß1 blocking antibody (Ha2/5). Addition of the antibody is indicated by `+', absence by `–'. The lower panel shows expression of total MAPK (ERK1 and ERK2), confirming equivalent levels in the different lysates. (B-E) ß1 Staining of 14 µm cryostat sections of floxed ß1/wt (B,C) and floxed ß1/null (D,E) neurospheres exposed to cre recombinase. Note that cells at the edge of the sphere still express high levels of ß1 in the floxed ß1/wt sphere (C, arrow) in contrast to the floxed ß1/null spheres (D,E). Note also that the negative controls (omitting the anti-ß1 antibody) show no staining (data not shown). The weaker labelling in the center of the spheres therefore most likely represents low levels of ß1 expression on more differentiated and non-mitotic cells, which is also reduced in the floxed ß1/null neurospheres exposed to cre recombinase. The decrease in ß1 expression was confirmed in the neurosphere cells by flow cytometry (F,G), which reveals a shift to the left (decrease) of the ß1 levels in the floxed ß1/null spheres (G) when compared with the floxed ß1/wt (F), following cre exposure. (H) Western blot of lysates from floxed ß1/wt (left) and floxed ß1/null (right) neurospheres showing the decrease of ß1 in the null cells (top lanes). The middle panels show levels of actin, confirming equal loading, whereas the lower panels show expression of ß-galactosidase, thus confirming excision of the floxed allele as discussed in the text. (I) In cre-exposed neurospheres, MAPK phosphorylation is considerably reduced in the floxed ß1/null (right) neurospheres, whereas total MAPK is maintained. After several passages, however, MAPK phosphorylation is the same in the floxed ß1/wt (left) and floxed ß1/null (right) neurospheres (J). WT, floxed ß1/wt; Null, floxed ß1/null. Scale bar: 20 µm for B-E.





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