Figure 7.

Differential activation and deactivation of ERK2 during HeLa cell attachment and spreading on gelatin plus and minus extracellular Ca²⁺. (A) Mobility shift assay. HeLa cells were washed twice in PBS-Mg plus or minus calcium and plated onto 60-mm gelatinized dishes. At the indicated times the excess medium was removed, and cells were washed twice with PBS-Mg containing 2 mM EGTA. After scraping, cells were lysed by the addition of 2× sample buffer. Sixty micrograms of protein from total cell lysates were loaded per well, and the electroblots were probed with mouse anti-human ERK2 IgG, The top band represents the phosphorylated form of ERK2. (B) Anti-phospho-ERK assay. HeLa cells were treated as described in A, except the electroblots were probed with a mouse anti-human phospho-ERK antibody. (C) Percent ERK2 activation plus (•) and minus (○) Ca²⁺ in A was quantified at each time point by densitometry. Data are expressed as a percentage of the total amount of ERK2 that becomes phosphorylated. A representative gel and scan from three independent experiments are shown.