Ab108980

Immunohistochemistry for TCF7L2 and EGLN2 was performed. Briefly, paraffin sections were baked for 60 min at 70 °C, de-paraffinized in xylene, rehydrated in gradually varied alcohol, and the sections were treated with 3% H₂O₂ to neutralize endogenous peroxidase for 30 min. The antigen retrieval was carried out with citrate buffer (pH = 6.0) in a pressure cooker. After antigen retrieval, the sections were incubated with primary antibody and secondary antibody. TCF7L2 antibody (Abcam, ab76151) was used at a dilution of 1:100. EGLN2 antibody (Abcam, ab108980) was used at a dilution of 1:50. The sections were then stained with DAB (3,3-diaminobenzidine) and terminated in PBS, and then counterstained with haematoxylin. Based on the staining intensity of TCF7L2 in each case, the sections were scored as follows: 0, negative; 1, weak; 2, moderate; 3, strong. Two observers graded the score of staining intensity independently.

Total cellular protein was extracted using RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with the Protease and Phosphatase Inhibitor Cocktail (Roche Applied Science). For the placenta, pieces were dissected as described for RNA preparation but were snap frozen in liquid nitrogen and stored at −80 °C. Frozen tissues were thawed for a few minutes in pre-chilled RIPA buffer (1 ml per100 mg), homogenized with a Polytron (Kinematica), and sonicated twice for 1 min (pulsed 2 s on/2 s off). After centrifugation (13 000 rpm., 15 min), 20–40 μg aliquots of protein were separated on MiniProtean TGX precast gels (Biorad) and transferred to a nitrocellulose membrane (Protran, Whatman). The membrane was blocked for 1 h at room temperature(RT) in PBS containing 0.05% tween-20 and 5% non-fat dry milk, incubated overnight at 4 °C with primary antibodies, and for 1 h at RT with an HRP-conjugated secondary antibody. Fractions were detected by Western Lightning Plus ECL (Perkin Elmer, Waltham, MA, USA). This study used primary antibodies against EPAS1/HIF2A (NB 100-122), FIH1/HIF1AN (EPR3658, NBP1-40688), TBP (NB 500-700), and MUC1 (EP1024Y, NB110-57234) from Novus Biologicals (Littleton, CO, USA). ARNT (ab2771), EGLN2 (ab108980), and MUC1 (ab101352) were purchased from Abcam (Cambridge, MA, USA).

After treatments, the cells were washed with PBS and proteins were extracted in 50 μl of lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 10% glycerol, and 1% NP-40) supplemented with 10 mM NaF, 1 mM Na₃VO₄, 10 μg/ml leupeptine, 1 μg/ml pepstatin and aprotinin, and 1 μl/ml PMSF saturated. Seventy micrograms of protein were boiled at 95 °C in Laemmli buffer and electrophoresed in 10% SDS/PAGE gels. Separated proteins were transferred to PVDF membranes (20 V for 30 min) and blocked in TBS solution containing 0.1% Tween 20 and 5% non-fat dry milk for 1 h at room temperature. Immunodetection of specific proteins was carried out by incubation with primary antibody against human HIF-1α (1:1000 dilution, #610,959, BD Biosciences, San Jose, CA, USA), murine HIF-1α (1:1000 dilution, #ab179483, Abcam, Cambridge, UK), PHD1 (1:1000 dilution, #ab108980, Abcam), PHD2 (1:1000 dilution, #ab109088, Abcam), PHD3 (1:1000 dilution, #ab30782, Abcam), OH-HIF-1α (1:1000 dilution, #3434S, Cell Signaling, Danvers, MA, USA), B55α (1:1000 dilution, #5689S, Cell Signaling), anti-HA (1:1000 dilution, clone 3F10 Roche), anti-Phospho-PHD2 Ser-125 (1:500) [13 (link)], and β-actin (1:10.000 dilution, #A5316, Merk, St Louis, MO, USA) overnight at 4 °C. After washing membranes, horseradish peroxidase-conjugated secondary antibody was added and detected by chemiluminescence system (GE Healthcare Europe GmbH).

Western blot was performed as previously described [19 (link)]. Briefly, after treatments, the cells were washed with PBS and proteins extracted in 50 μL of lysis buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10% glycerol, and 1% NP-40) supplemented with 10 mM NaF, 1 mM Na3VO4, 10 μg/mL leupeptin, 1 μg/mL pepstatin and aprotinin, and 1 μL/mL PMSF saturated. Thirty micrograms of proteins were boiled at 95 °C in Laemmli buffer and electrophoresed in 10% SDS/PAGE gels. Immunodetection of specific proteins was carried out by incubation with primary antibody against HIF-1α (1:1000 dilution, #610,959, BD Biosciences, San Jose, CA, USA), OH-HIF-1α (1:1000 dilution, #3434S, Cell Signaling, Danvers, MA, USA), B55α (1:1000 dilution, #5689S, Cell Signaling), anti-HA (1:1000 dilution, #clone 3F10 Roche), anti-Phospho-PHD2 Ser125 (1:500 dilution) [10 (link)], anti-PHD1 (1:1000 dilution, #ab108980, Abcam), anti-PHD2 (1:1000 dilution, #ab109088, Abcam), PHD3 (1:1000 dilution, #ab30782, Abcam) and β-actin (1:10.000 dilution, #ab49900, Abcam, Cambridge, UK), overnight at 4 °C. After washing membranes, horseradish peroxidase-conjugated secondary antibody was added and detected by chemiluminescence system (GE Healthcare Europe GmbH).