HER1 Signaling Mediates Extravillous Trophoblast Differentiation in Humans: Western Blot Analysis

Western Blot Analysis
Serum-starved JAR cells were stimulated with either EGF or HBEGF (10 ng/ml) over a 4-h time course. Following treatment, cells were washed with ice-cold PBS and lysed using a Bio-Rad cell lysis kit according to the manufacturer’s instructions (Bio-Rad, Mississauga, ON). Equal amounts of JAR cell proteins per lane were analyzed by Western blot analysis. Briefly, 60 ig of total protein per sample were added to NUPAGE LDS sample buffer (Invitrogen) with 2.5% p-mercaptoethanol and boiled for 5 min. Proteins were run on precast 3%—8% Tris-Glycein gels (Invitrogen) and transferred to polyvinylidene fluoride membrane (Millipore) at 4°C overnight. Membranes were blocked with 5% nonfat milk in Tris-buffered saline with Tween (TBS-T; 10 mM Tris [pH 7.5], 100 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature. Membranes were incubated with either rabbit polyclonal anti-phospho HER1, Tyr992, Tyr1045, Tyr1068, or anti-phospho p85 PIK3 (New England Biolabs, ON) at 4°C overnight, and then washed and incubated with anti-rabbit horseradish peroxidase (1:2000)-linked secondary antibody (New England Biolabs) for 1 h at room temperature. Membranes were then stripped and reprobed with antibodies against the respective nonphosphorylated antibody (anti-HER1 and Anti p85 PIK3, 1:1000; New England Biolabs). To further assess equal loading of protein samples, blots were stripped for a second time and reprobed with an antibody against the housekeeping protein p-actin (Abcam, Cambridge, MA). Antibody reactions were detected using Bio-Rad WesternC chemiluminescence detection kit (Bio-Rad), followed by detection of chemiluminescence and band analysis using the VersaDoc 6 gel documentation system and Quantity One Software (Bio-Rad).
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Bioplex ELISA
Lysates from JAR cells stimulated with either EGF or HBEGF were prepared as above in Bio-Rad cell lysis buffer and diluted to 0.7 ig/ml. To detect downstream HER1 targets we performed a multiplex phosphoprotein assay for five proteins (MAP2K1, also known as MEK1; Ser217/Ser221), extracellular signal-regulated kinase 1 and 2 (MAPK3/1, also known as ERK1/ 2; Thr202/Tyr204, Thr185/Tyr187), 90-kDa ribosomal S6 kinase (RPS6KA, also known as p90RSK; Thr359/Ser363), protein kinase B (AKT1, also known as [PKB]/Akt; Ser473), and glycogen synthase kinase 3 (GSK3A/B; Ser21/ Sei9). A corresponding multiplex total protein assay for MAP2K1, MAPK3/1, RPS6KA, and AKT1 was performed on matching samples. Samples were run in duplicate and according to the manufacturer’s instructions (Bio-Rad). Sample data is presented as the median fluorescence intenstity of the phosphoprotein over the corresponding total protein and relative to the time control for each experiment.
Statistical Analysis
Statistical analysis of data was performed using Prism software on normally distributed data using a one-way or two-way ANOVA where appropriate with either a Dunnet or Bonferroni multiple-comparison test respectively. Error bars represent the SD of the mean of three independent experiments performed in triplicate. P < 0.05as compared with respective controls are considered significant.

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