GPER/GPR30 expression in normal lung cell lines and lung adenocarcinoma cell lines. A) The expression of GPER was determined by realtime quantitative PCR in each of the normal lung cell lines (open bars), lung adenocarcinoma cell lines (grey bars), and MCF-7 human breast cancer cells (black bar). Values are the average of 4–6 biological replicates ± SEM. B-D) Representative western blot analysis of GPER/GPR30 expression in the indicted cell lines. 30 μg of whole cell extract (WCE) were separated on 10% SDS PAGE gels with MW marker migration indicated at the left of each blot. The diagrams at the top of B-C, and D indicate the region of GPER recognized by the 3 different polyclonal antibodies used: B) Novus NBP-1-31239, C) Novus NLS4271; D) Santa Cruz sc-4854-R. The MW of GPR30 is estimated to be 42 kDa, but higher MW sizes have been reported due to glycosylation and interaction with other proteins. Bands are identified as glycosylated and nonglycosylated based on reports cited in the text, but could include interaction with other proteins. For each GPER western, the membrane was stripped and reprobed for β-actin as a loading control. Quantitation of GPER was evaluated by summing all immunoreactive bands and dividing by β-actin, then normalizing to HBEC2-KT in each blot. Panel E is a summary of all westerns (not all westerns are shown) with each antibody. For Westerns with NBP1-31239 and sc-48524, values are the mean ± SEM from 3 separate blots using different WCE.