Western blot analysis determines the responses of mitogen-activated protein kinases (MAPKs) after incubation in pH 6.0 or 7.4 in wild-type (WT), acid-sensing ion channel (ASIC3)-/- and ASIC1-/- fibroblast-like synoviocytes (FLS). (A) Representative western blots for extracellular signal-regulated kinase (ERK), p-ERK, p-p38, p-38, c-Jun N-terminal kinase (JNK), p-JNK and actin treated with pH 7.4 or pH 6.0. Blots were always run with pH 7.4 or pH 6.0 simultaneously. Notice the decreases in p-ERK with pH 6.0 when compared to pH 7.4. The bands that were quantified for each protein are highlighted with an arrow and are previously described by us
. (B) WT FLS: density of the individual bands was taken using Image J software and pH 6.0 was normalized to pH 7.4; pH 6.0 and pH 7.4 were always run simultaneously on the same blots to facilitate comparison between groups. Expression values of 1.0 represent no change, <1.0 represent a decrease in expression, and > 1.0 represent an increase in expression. p-ERK protein expression significantly decreased after exposure to pH 6.0 in WT FLS when compared to the response at pH 7.4 (*P <0.05). (C) ASIC3-/- FLS: p-ERK protein expression was significantly greater 15 minutes after exposure to pH 6.0 in ASIC3-/- FLS when compared to WT FLS (+
P <0.05). (D) ASIC1-/- FLS: p-ERK protein expression significantly decreased after exposure to pH 6 in ASIC1-/- FLS when compared to the response at pH 7.4 (*P <0.05), and was similar to the decreased expression observed in WT FLS.