D1d8q

Protein extracts, separated by SDS-PAGE and transferred onto NC membranes (Schleicher and Schuell, Einbeck, Germany), were probed with antibodies against GLUT1 (sc-377228, 1:1000, Santa Cruz, Heidelberg, Germany), OGA (JG40-05, 1:1000, Thermo Fisher, Branchburg, NJ, USA), OGT (D1D8Q, 1:1000, Cell Signaling Technology, Danvers, MA, USA), HIF-1α (GTX127309, 1:1000, GeneTex, Hsinchu, Taiwan), Hydroxy-HIF-1α (Pro564) (D43B5) Rabbit mAb (#3434, 1:1000, XP) or Anti-beta Actin antibody (mAbcam 8226, 1:1000, Abcam, Cambridge, UK) or proteins of interest were detected with peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG, Fc Fragment Specific (115-035-008, 1:1000, Jackson ImmunoResearch, West Grove, PA, USA) or Rabbit Anti-Goat IgG Antibody, HRP conjugate, (abbbit IgG, AP106P, 1:1000, Sigma-Aldrich) and visualized with the Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Merck, Rahway, NJ, USA), according to the protocol provided. The Western blotting was repeated three times and included in the statistics. Quantitation of bands was conducted using Image J.

Five microns sections from formalin fixed paraffin-embedded placenta tissues were de-paraffinized with xylenes and rehydrated through a graded alcohol series. Heat induced epitope retrieval (HIER) was performed by immersing the tissue sections in Target Retrieval Solution, Low pH (DAKO) in the PT Link (DAKO). Immunohistochemical staining was performed using a horseradish peroxidase labeled polymer (Agilent, K4003) according to manufacturer’s instructions. Briefly, slides were either treated with 3% hydrogen peroxide and 10% normal goat serum for 10 min each, and exposed to primary antibody for OGT (1:200, Cell Signaling, D1D8Q) for 1 h at room temperature. Slides were exposed to the appropriate HRP labeled polymer for 30 min and DAB chromagen (Dako) for 5 min. Slides were counterstained with Hematoxylin (Fisher, Harris Modified Hematoxylin), blued in 1% ammonium hydroxide, dehydrated, and mounted with Acrymount. Consecutive sections with the primary antibody omitted were used as negative controls.