Fig. 2

Validating the method for studying molar ratio of ASICs in the brain. a Specificity of an ASIC2 antibody. 3T3 cells transfected with ASIC2a or ASIC2b and brains from wild-type (WT) or ASIC2-/- mice were blotted with a rabbit ASIC2 IgG (red) and a mouse Tubulin (green) antibody, and detected simultaneously using a laser scanning imaging system (Li-Cor). Arrowheads on the right indicate the relative position of the indicated proteins. Note specific ASIC2a (~66KD) and ASIC2b (~75KD) bands were present in wild-type but not in ASIC2-/- brain lysates. b Diagram showing the ASIC1a-ASIC2a (1a-2a) fusion construct and the relative position of the region recognized by the antibodies used. c Blots showing the expression of the 1a-2a dimer and brain ASICs. Left three lanes were loaded with various amounts of lysate from cells overexpressing the 1a-2a fusion construct (runs at ~130KD). The blot was blotted for ASIC1a, ASIC2, and tubulin at the same time. d Quantification showing the density (left graph) of ASIC1a or 2a in (c) and the ratio (right graph) of ASIC1a:ASIC2 pixels with different levels of loading. e Summary data for the change of 1a:2 ratio obtained for the 1a-2a dimer at different loading. In order to compare between experiments, the ratio at the lowest loading was arbitrarily set to 1 in each experiment. Note that when we calibrated 1a/2 ratio for the brain proteins, we used the raw ratio obtained in panel G as the calibrator (see Results for further elaboration). (f & g) Blots (representative from 4 repeats) and quantification showing the signals of ASIC1a, 2a, and 2b at loadings between 4 and 64 μg of total brain protein