Blocking β1 integrin dampens activation of pY1214 and P38 of matrix-bound VEGF₁₆₅. (A) Confluent HUVEC monolayers were starved for 6 h and pretreated with β1-inhibiting antibodies for 15 min, then treated with the indicated reagents for an additional 15 min. Cell lysates were collected, resolved on 4–12% SDS-PAGE, probed with phopho-p38 antibody, and reprobed with total p38 antibody. (B) Same as A, treated with indicated reagents for 15 min and probed with antibody against pY1214. Membranes were then stripped off and reprobed with total VEGFR2 antibody. (C) HUVECs were treated as in A. Cell lysates were collected and immunoprecipiated with VEGFR2 antibodies. Immunoprecipitated proteins were probed with anti-β1 integrin or anti-VEGFR2. The top panel shows cells without preincubation with β1-inhibiting antibodies. The bottom panel shows cells preincubated with β1-inhibiting antibodies. (D) Mouse liver ECs from β1^fl/fl mice were isolated and treated with adeno-Cre virus to induce deletion of the gene. After 3 d, cultures were treated with indicated growth factors for 15 min and cell lysates were probed with pY1214, GAPDH (36 kD), and β1 antibodies. Blots were then stripped and reprobed with anti-Cre (35 kD). (E) Similar experimental setting as D but probed with pY1175. (F) Confluent β1^fl/fl or β1^ko MLEC monolayers in 96-well plates were starved overnight and exposed to the indicated treatments for 5, 15, or 30 min. p38 activation was determined by cell-based ELISA and the fold change at each time point was calculated by phospho-p38 over total p38 compared with no treatment. The difference between pP38 activation in WT and KO cells is statistically significant at 15 and 30 min. *, P < 0.05 (n = 4).