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. 2019 Aug 21;160(11):2692–2708. doi: 10.1210/en.2019-00177

Figure 2.

Figure 2.

Membrane-bound ER initiates phosphorylation of several kinases downstream upon E2 exposure. (A) Day 7 prostaspheres (PSs) treated with vehicle or 10 nM E2 for 15, 30, and 60 min were subjected to phosphokinase arrays spotted with 43 different kinase proteins. The table shows 18 proteins that exhibited >50% increased phosphorylation levels over vehicle with E2 exposure during the course of 60 min. Values are an average of n = 3 repeats relative to vehicle (normalized to 1) using ImageJ image analysis software. (B–D) Western blot analysis shows (B) phosphorylation MAPK pathway molecules p38, ERK1/2, and JNK, (C) AKT pathway cascades including SRC, AKT, NF-κB, and ERα S167, and (D) membrane-bound receptors IGF1R and EGFR during a 15-, 30-, and 60-min time course following E2 exposure to day 7 PSs (n = 3). (E) Western blot analysis of day 7 PSs treated with vehicle and 10 nM E2 with/without 1 μM ICI for 30 min shows an attenuation of E2-mediated phosphorylation of both AKT and ERK with ER antagonism. Graphic representation is shown at right for n = 4. *P < 0.05 vs vehicle and E2 plus ICI. (F) Day 7 PSs treated with 10 µM E2 dendrimer (impermeable to nuclear membrane) show a marked increase in phosphorylation of AKT and ERK1/2 by the E2 dendrimer, comparable to 10 nM E2. Graphic representation at right for n = 4.