Ab32551

SDS-PAGE was carried out on tissue homogenates with each sample standardized to 1.5 μg DNA per well. Proteins were separated on 4%–12% Bis-Tris Plus SDS gels and transferred to 0.45 μm polyvinylidene difluoride membranes (Life Technologies, Carlsbad, USA). Membranes were incubated overnight at 4°C with primary antibodies against carbonic anhydrase IX (CA-IX) (1/200, R&D Systems, AF2188), glucose transporter 1 (GLUT1) (1/1,000; Abcam, ab32551), phosphorylated histone protein γH2AX (1/2000; Abcam, ab11174), or ß-actin (1/10,000, Sigma, A5316), and for 1 h at room temperature with secondary horseradish peroxidase-conjugated antibodies (1/5,000, DAKO). β-Actin was used as loading control. For GLUT1, the same positive control (20 µg protein of 1% O₂ treated T24 cell lysate) was run on each gel to normalize signals between the blots. For CA-IX, a clear cell renal cell carcinoma tissue sample (University of Otago Human Ethics committee H14/020 (37 (link)), adjusted to 1.5 μg DNA per well, was similarly run on each gel. For γH2AX, a positive control (WiDr cells from ATCC treated for 4 h with 20 mM ascorbate, 20 µg protein) was run on each gel. Protein bands were visualized using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, USA), and quantified with the Alliance 4.7 imaging system and the ImageJ software (36 (link), 37 (link)).

The expression of GLUT1 and CAT3 protein was analyzed in placental homogenates. Three placentae from 3 different dams in each group were harvested and processed. The placentae were homogenized and suspended in a lysis buffer (BioDev-Technology, Beijing, China). Twenty μg of protein were resolved by SDS-PAGE (12% gel) and then transferred to polyvinylidene difluoride membrane. After blocking in blocking solution [Tris-buffered saline with 0.1%Tween 20 (TBST) and 5% BSA] for 1.5 h, the membranes were incubated at 4 °C overnight with specific antibodies anti-GLUT1 (1:1000 dilution; ab32551, Abcam) and anti-CAT3 (1:1000 dilution; ab113985, Abcam) respectively in 5% BSA. The membrane was then washed 3 times with TBST, and bands were visualized with the appropriate horseradish peroxidase-conjugated secondary antibodies and the enhanced chemiluminescence system, in accordance with the manufacturer’s instructions.

Cells were harvested in protein lysis buffer consisting of 25 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 0.5% Triton-X100, 0.5% NP-40, 1x proteinase inhibitor cocktail (PIC) (Sigma), 0.5 mM Na₃VO₄, 10 mM NaF and 10 mM C₃H₇Na₂O₆P. Protein concentration was quantified using the BCA Protein Assay (Pierce) and 20 µg of protein were loaded to 10% SDS-polyacrylamide gels and blotted to nitrocellulose membranes. The antibodies used for Western Blot were specific for thymoma viral proto-oncogene 1 (AKT), p-AKT (9272 and 9271, Cell Signaling), GLUT-1 (abcam ab32551), and VCP (ab11433, Abcam) for normalizing. Quantification was performed using the ImageJ software.