(A) Proportional diagrams illustrating the number of specifically enriched and identified phosphoproteins, basally occurring, and induced by MeCh stimulation (10 nM to 100 µM) of control or CMP-treated cells. MeCh-induced protein phosphorylation in diverse cellular contexts. Each experimental point on the curves (fitted using sigmoidal dose-response functions in GraphPad Prism) represents the mean ± SEM of three independent experiments. The maximal response for MeCh in each cellular context (control versus CMP) was considered to be 100% in each circumstance. (G) Response-normalized MeCh ERK1/2 log dose-response curves. (F) Log dose-response curves for acetyl-β-methycholine (MeCh)-mediated ERK1/2 activation in control (black circles) or CMP-treated (white square) cells. Data is represented as mean ± standard error of mean (SEM). Quantification was performed using ImageQuant 5.2 and statistical analysis was performed on three independent experiments using GraphPad Prism version 5.02 with a Student’s t-test (p<0.05, * p<0.01, ** p<0.001, ***).

Quantification was achieved as follows: western blot relative absorbance units (x1000) minus background absorbance subtraction per square pixel: ((AU-B)/px 2). Quantification of CMP-mediated expression changes of LMNA (B), GIT2 (C), CALM1 (D) and CALR (E). (A) Representative western immunoblot (IB) analysis of control (PBS-vehicle treated) or CMP treatment (7 days, 10 nM hydrogen peroxide treatment) effects upon cellular expression of lamin A (LMNA), G protein-coupled receptor kinase interacting ArfGAP 2 (GIT2), calreticulin (CALR) and calmodulin (CALM1). Chronic minimal peroxide-mediated alteration of protein expression and ligand responses.

Venn diagram graphpad prism