C300 imager

Denatured samples were loaded on a 12% acrylamide sodium dodecyl sulphate–polyacrylamide electrophoresis (SDS-PAGE) gel, prepared as previously reported (Sattlegger et al., 2013 (link)). After gel electrophoresis in 1 × Tris–glycine buffer, proteins were transferred to a 0.22 μm PVDF via the semi-dry transfer method at 20 V for 20 min (Trans-Blot^® SD Semi-Dry Transfer Cell, Bio-rad), using 1 × Tris glycine buffer containing 20% methanol.
Western blotting assay was performed as previously described (Sattlegger et al., 2013 (link)). Primary antibodies were directed against the FLAG tag (FLAG-tag rabbit polyclonal antibody, 1:5,000, Huaxingbio, No. HX1819). The secondary antibody containing horseradish peroxidase conjugated to Goat anti-Rabbit antibodies (Huaxingbio, No. HX2031) was incubated with a dilution of 1:5,000. The horseradish peroxidase was visualized using an ECL chemiluminescence detection reagent (Super ECL, No. HXP1868, Huaxingbio) as described by the manufacturers, and imaged with a C300 imager (Azure Biosystems, Inc., Dublin).

RNA isolation, cDNA synthesis, and qPCR were performed as previously described [25 (link)]. The following PCR primers were used: PEPT1/SLC15A1 forward: 5′-CTCCCAATGTTCTGGGCCTT-3′ and reverse: 5′-CGTTCACGGTCTGCATCTGA-3′; PEPT2/SLC15A2 forward: 5′-ATCAGCAGGGTTCACGATGG-3′ and reverse: 5′-CCACACTTGGAGACCAGACG-3′. Cell line and PDX protein lysates were prepared, and western blot analysis was performed, as previously described [25 (link)]. In short, 30 µg of protein was loaded per lane in each of the western blots and electrophoretically separated on 10% SDS–Page gels. All proteins were transferred at 4°C overnight at 20 V to a nitrocellulose membrane (Bio-Rad), blocked with 5% Blotting Grade Blocker (Bio-Rad) at room temperature for 90 min, then incubated with primary antibody at 4°C overnight. All primary antibodies were diluted 1000-fold, with the exception of anti-β-actin, which was diluted 10 000-fold. After washing with TBST, the membrane was incubated for 1 h at room temperature with correlating secondary antibodies at a dilution of 3000-fold, washed in TBST again, then incubated for 5 min in Pierce ECL solution (ThermoFisher), before being visualized with either autoradiography films or an Azure Biosystems c300 imager.

Aliquots of whole-cell lysates were prepared in NP-40 buffer (150 mM NaCl, 1% NP-40, and 50 mM Tris-HCl, pH 8.0) and fractionated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were transferred from the SDS gel to a nitrocellulose membrane for 10 min by using a Trans-blot Turbo (Bio-Rad). The nitrocellulose membrane was blocked in 5% milk (% w/v) in Tris-buffered saline and 0.1% Tween-20 (TBS-T) for 30 min and incubated overnight with primary antibodies (1:1000) against HIF-1α (#610959, BD Biosciences) and GFP (#ab13970, Abcam). After washing three times with TBS-T, the nitrocellulose membrane was incubated with the corresponding anti-mouse (1:2500) (#AC2115, Azure Biosystems) or anti-chicken (1:5000) (AP162P, Sigma-Aldrich) HRP-secondary antibody for 1.5 h at room temperature with orbital shaking. β-actin was detected with a β-actin HRP-conjugated antibody (1:10,000) (#HRP-60008, Proteintech). ECL (Enhanced Chemiluminescent Substrate) (Perkin Elmer) was utilized as the substrate for HRP-catalyzed detection. The chemiluminescent signal was imaged by using a c300 imager (Azure Biosystems). DsRed-only and GFP-only cell lines were used as control for the GFP antibody.