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Figure 4 | Breast Cancer Research

Figure 4

From: Membrane estrogen receptor-α levels predict estrogen-induced ERK1/2 activation in MCF-7 cells

Figure 4

Optimization of 96-well plate immunoassay for membrane estrogen receptor (mER) quantification in MCF-7 cells. (a) Different concentrations of estrogen receptor-α-specific C-542 antibody (1–12 μg/ml) were tested. Enzymatic paranitrophenol phosphate (pNpp) hydrolysis yielding the paranitrophenol (pNp) product was monitored at 37°C for 5, 15 and 30 min, as shown. Cells pretreated for 72 hours with medium containing dextran-coated charcoal-stripped serum (DCSS) versus defined medium (DM) are represented with closed and open circles, respectively. (b) Antibody binding compared in nonpermeabilized (open bars) and permeabilized (shaded bars) conditions. no1°no2° represents primary and secondary antibodies omitted; no1° represents primary antibody omitted (only secondary antibody applied); mIgG1k represents mouse IgG1k isotype control; peptide comp. represents C-542 epitope peptide blocking of the interaction with primary antibody; anti-clathrin represents our test for cell membrane integrity. Values are expressed as means ± standard error. CV, crystal violet.

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