Specificity of the effect of K⁺-channel blockade on ERK-2 activation. (A) Direct effect of 4-AP on ERK-2 activity. ERK-2 was immunoprecipitated and measured for activity in the presence and absence of 3 mmol/L 4-AP in kinase buffer. (B) Effects of different K⁺-channel blockers on EGF-induced ERK-2 activation. Serum-starved ML-1 cells were either untreated or were stimulated with EGF (50 ng/mL) for 15 minutes in the presence or absence of 4-AP (2 mmol/L), TEA (10 mmol/L), or Ba²⁺ (5 mmol/L). The cells were collected at the end of the treatment, and ERK-2 activities were measured. (C) Effect of high extracellular K⁺ on EGF- and FBS-induced ERK-2 activation. Serum-starved ML-1 cells were either untreated or were stimulated with EGF (50 ng/mL) or FBS (10%) for 15 minutes in normal medium or in medium containing high K⁺ (60 mmol/L). The cells were then collected and assayed for ERK-2 activity. (D) Effect of 4-AP on TPA-induced ERK-2 activation. Serum-starved ML-1 cells were untreated or treated with TPA (1 nmol/L) for 10 or 30 minutes in the presence or absence of 4-AP. At the end of the treatment, cells were collected and measured for ERK-2 activity. (E) Effect of 4-AP on high-osmolarity–induced ERK-2 activation. Serum-starved ML-1 cells were untreated or treated with high-osmolarity medium (600 mmol/L sorbitol) in the presence or absence of 2 mmol/L 4-AP. The cells were then collected and measured for ERK-2 activity. (F) Effect of extracellular Ca²⁺ on EGF-induced ERK-2 activation and on the inhibition of ERK-2 activation by K⁺-channel blockade. Serum-starved ML-1 cells were untreated, treated with EGTA alone, or treated with EGTA plus 4-AP (2 mmol/L) for 30 minutes. Cells were then left untreated or stimulated with EGF (50 ng/mL) for 15 minutes. The cells were collected at the end of the treatment and measured for ERK-2 activity and protein levels.