FAK and MAPK (ERKs) activation after integrin-mediated adhesion.
Factor-starved G1E-ER2 cells were cultured on FN peptides in the presence of SCF and analyzed at the indicated time points. (A) Cell lysates were collected and subjected to Western blot analysis with an anti-FAK antibody. Bottom panels indicate the position of phosphorylated (pFAK) and unphosphorylated (FAK) FAK. Upper panels (bars) demonstrate the relative phosphorylation of FAK; 48 hours taken as 100. Bars denote the mean relative phosphorylation (± SEM) of 3 different experiments. Asterisk indicates P < .05 α5β1 (CH271) versus α4β1 (H296). (B,C) Factor-starved G1E-ER2 cells were left unstimulated or cultured on BSA (B) or on indicated FN peptides mediating adhesion via α4β1 (H296) and α5β1 (CH271) (C) in the presence of SCF. Cell lysates were collected and subjected to Western blot analysis with a rabbit antiphospho-ERK antibody that specifically detects phosphorylated T202 and Y204. Bottom panels show total Erk in each lane. The positions of the phosphorylated ERK-1 (pErk-1) and ERK-2 (pErk2) are indicated. Upper panels (bars) demonstrate the relative phosphorylation of ERK-1 and ERK-2 at residues T202 and Y204. Data are presented relative to the phosphorylation of ERK-1 and ERK-2 after 10 minutes (B) (with the level at 10 minutes taken as 100) and 120 minutes (C) (with the level at 120 minutes taken as 100). Bars denote the mean relative phosphorylation (± SEM) of 3 different experiments. Asterisk indicates P < .05 α5β1(CH271) versus α4β1 (H296). (D) G1E-ER2 erythroid progenitors were cultured on BSA or on FN peptides H296 or CH271 in the presence of SCF and the MEK inhibitor (PD98059). Proliferation was measured by thymidine incorporation assay. Bars denote the inhibition in proliferation (± SEM) of 3 independent experiments performed in replicates of 6. Asterisk indicatesP < .05 α4β1 (H296) versus α5β1 (CH271) and BSA; and double asterisks indicate P < .05 α4β1 (H296) and α5β1 (CH271) versus BSA.
