Protein expression of total and phosphorylated S6K1 (Thr-389), 4E-BP1 (Thr-37/46 or Thr-70), S6 ribosomal protein (Ser-235/236), Akt (Thr-308), STAT3 (Tyr-705), and AMPKα (Thr-172) was determined in placental homogenates using commercial antibodies (Cell Signaling Technology, Boston, MA). Protein expression of the system A amino acid transporter isoforms (SNAT) 1, 2, and 4 and the system L amino acid transporter isoforms LAT1, LAT2, and 4F2hc was analyzed in placental MVM preparations. The justification for determining protein expression of transporters in MVM rather than in homogenates is that trophoblast nutrient transporters mediate cellular uptake and transfer across the placental barrier only if localized in the syncytiotrophoblast plasma membranes. Thus, data on amino acid transporter protein expression in MVM is much more informative than determination of protein expression in placental homogenates. The SNAT1 antibody was received as a generous gift from Jean Jiang (University of Texas Health Science Center San Antonio). A polyclonal SNAT2 antibody was generated in rabbits (43 (link)). Affinity-purified polyclonal anti-SNAT4 antibodies were produced in rabbits using the epitope YGEVEDELLHAYSKV in human SNAT4 (Eurogentec, Seraing, Belgium). Antibodies targeting the LAT1 and LAT2 were produced in rabbits as described previously (44 (link)). The 4F2hc antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-β actin was from Sigma-Aldrich (St. Louis, MO).
Western blot analysis was performed as previously described (31 (link)). In brief, 20 μg of total protein were loaded onto a SDS-PAGE gel (7% for S6K1, Akt, STAT3, AMPKα, SNAT 1/2/4, and 4F2hc; 12% for 4E-BP1; and 4–12% for LAT1 and LAT2), and electrophoresis was performed at a constant 100 V for 2 h. Proteins were transferred onto nitrocellulose membranes overnight at a constant 30 V. After the transfer, the membranes were blocked in 5% milk in Tris-buffered saline (wt/vol) plus 0.1% Tween 20 (vol/vol) for 1 h at room temperature. Membranes were incubated with primary antibodies overnight at 4 C. Subsequently, membranes were incubated with the appropriate secondary peroxidase-labeled antibodies for 1 h. After washing, bands were visualized using enhanced chemiluminescence detection reagents (GE Healthcare, Chalfont St. Giles, UK). Blots were stripped using β-mercaptoethanol as described previously (45 (link)) and reprobed for β-actin as a loading control. Analysis of the blots was performed by densitometry using an AlphaImager (Alpha Innotech Corp., San Leandro, CA). For each protein target and gestational age, the mean density of the C sample bands was assigned an arbitrary value of 1. Subsequently, all individual C and LP density values at a particular gestational age were expressed relative to this mean.
Western blot analysis was performed as previously described (31 (link)). In brief, 20 μg of total protein were loaded onto a SDS-PAGE gel (7% for S6K1, Akt, STAT3, AMPKα, SNAT 1/2/4, and 4F2hc; 12% for 4E-BP1; and 4–12% for LAT1 and LAT2), and electrophoresis was performed at a constant 100 V for 2 h. Proteins were transferred onto nitrocellulose membranes overnight at a constant 30 V. After the transfer, the membranes were blocked in 5% milk in Tris-buffered saline (wt/vol) plus 0.1% Tween 20 (vol/vol) for 1 h at room temperature. Membranes were incubated with primary antibodies overnight at 4 C. Subsequently, membranes were incubated with the appropriate secondary peroxidase-labeled antibodies for 1 h. After washing, bands were visualized using enhanced chemiluminescence detection reagents (GE Healthcare, Chalfont St. Giles, UK). Blots were stripped using β-mercaptoethanol as described previously (45 (link)) and reprobed for β-actin as a loading control. Analysis of the blots was performed by densitometry using an AlphaImager (Alpha Innotech Corp., San Leandro, CA). For each protein target and gestational age, the mean density of the C sample bands was assigned an arbitrary value of 1. Subsequently, all individual C and LP density values at a particular gestational age were expressed relative to this mean.
