Laemmli buffer

For each sample, 5 million collected cells were resuspended in 500 μl IPH buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40 and 10% glycerol supplemented with 1× Protease/Phosphatase inhibitors (Thermo Fisher Scientific, 78444), 0.1 mM PMSF) before sonication in a QSONICA 800R instrument (30 s on and 30 s off, 15 min in total, 4 °C). Protein supernatant was then collected after centrifugation for 15 min at 18,400g and 4 °C. The proteins were incubated overnight at 4 °C with 2 μg of the indicated antibodies (listed in Extended Data Table 1) in the presence of protein G Dynabeads (Life Technologies, 10004D) and 100 mg ml⁻¹ Ethidium Bromide (Invitrogen, 15585-011). Beads were washed five times with IPH buffer and eluted by boiling in 2× Laemmli buffer (Boston BioProducts, BP-111R).

Tryptic digestion of purified rhNQO1 was performed in 25mM Tris-HCl, pH 7.4 containing 5μM FAD. Reactions (50μl) contained 2μg rhNQO1 in the absence and presence of 0.2mM NADH. Proteolytic digestion was initiated by the addition of 0.1μg of TPCK-treated trypsin (bovine pancreas, Sigma-Aldrich) for 30 min at 37°C after which 2μl of the above reaction was diluted in 278μl of 3X Laemmli buffer (Boston BioProducts, Ashland, MA), heated to 85°C for 7 min and 20μl was separated by 12% SDS-PAGE (precast Mini-Protean, BioRad, Hercules CA). Proteins were then transferred to a 0.2μm PVDF membrane in 25mM Tris containing 192mM glycine and 20% (v/v) methanol at 150 volts for 2 h at 4°C. Membranes were blocked in 10mM Tris-HCl, pH 7.8, 150mM NaCl and 0.2% (v/v) Tween-20 (TBST) containing 5% (w/v) non-fat dry milk for 1h at 22°C and primary antibodies were diluted in TBST containing 5% non-fat dry milk (see S1 Table, for primary antibody dilutions), and then used to probe membranes for 1h at 22°C. Membranes were washed extensively in TBST followed by the addition of horseradish peroxidase-conjugated secondary antibodies diluted in TBST containing 5% non-fat dry milk for 30 min at 22°C. Membranes were washed extensively with TBST and protein bands were visualized using enhanced chemiluminescence (GE Healthcare Life Sciences, Pittsburgh, PA).

Purified rhNQO1 (0.2μg) was added to 25mM Tris-HCl, pH 7.4 containing 1mg/ml BSA and 5μM FAD (0.5ml final volume) in the presence or absence of 0.2mM reduced pyridine nucleotides or analogs. After 15 min either anti-NQO1 A180 or C-term antibody (2μg) was added for 1 h at 22°C followed by 25μl of protein A/G plus agarose (Santa Cruz Biotechnology) for an additional 30 min. Beads were collected by centrifugation at 5000 rpm for 1 min at 22°C, washed 3 times with 0.5ml of 25mM Tris-HCl, pH 7.4 containing 1mg/ml BSA and 5μM FAD, then once with 0.5ml of 25mM Tris-HCl, pH 7.4. Beads were resuspended in 25μl of 3X Laemmli buffer (Boston BioProducts), heated to 85°C for 7 min and 20μl was analyzed by immunoblot analysis as described above (see Tryptic Digestion of NQO1).

After overnight starvation, cells were treated with basal medium, basal medium + VEGF121 (20 ng/mL), or VEGF165 (10 ng/mL). Treatment was allowed to incubate at 37°C for 0, 5, 10, 15, 30 minutes, and 1, 2, 4 h for VEGF121, or the standard 5 mins for basal medium (negative control) and VEGF165 (positive control). The media was then removed, and cells were lysed on ice with RIPA buffer containing protease and phosphatase inhibitors (Thermo Fischer Scientific, Waltham, MA) then centrifuged. The supernatant was then resuspended in 1X Laemmli buffer (Boston Bio Products, Ashland, MA) and analyzed for phosphorylated and total VEGFR2 (P-Y1175-VEGFR2, tVEGFR2) with Western immunoblot.

After overnight starvation, cells were treated with basal medium, basal medium + increasing doses of VEGF121 (2.5, 5, 10, 20, 100, 200 ng/mL), or VEGF165 (10 ng/mL) as a positive control for five minutes at 37 °C. The media was removed, and cells were lysed on ice with RIPA buffer containing protease and phosphatase inhibitors (Thermo Fischer Scientific, Waltham, MA) then centrifuged. The supernatant was then resuspended in 1X Laemmli buffer (Boston Bio Products, Ashland, MA) and analyzed for phosphorylated and total VEGFR2 (P-Y1175-VEGFR2, tVEGFR2) with Western immunoblot.

Proteins were eluted from beads by heating at 95 °C for 5 min in 100 μL 1x Laemmli buffer (Boston Bioproducts), followed by separation in a 4–12% Bis-Tris Deep Well gel and visualization by Coomassie staining. Each gel lane was then collected and cut into 12 equal-volume gel slices, which were subject to in-gel trypsin digestion as described before^{80 (link)}. In brief, gel segments were destained (in 50 mM ammonium bicarbonate buffer with 50% methanol) first, followed by reduction (in 10 mM TCEP), alkylation (in 50 mM iodoacetamide), dehydration (in acetonitrile) and finally digestion at 37 °C for 12–16 h in 50 mM ammonium bicarbonate buffer containing 100 ng porcine sequencing grade modified trypsin (Promega). Tryptic peptide products were then acidified (in 0.1% formic acid) and separated by an in-line 150 × 0.075 mm column loaded with reverse phase XSelect CSH C18 2.5 um resin (Waters) using a nanoAcquity UPLC system (Waters). Eluted peptides were ionized by electrospray (2.15 kV) and analyzed using an Orbitrap Fusion Tribrid mass spectrometer (Thermo), with MS data and MS/MS data acquired by the FTMS (profile mode; with resolution of 240,000 and range from 375 to 1500 m/z) and ion trap (centroid mode; with normal mass range and precursor mass-dependent normalized collision energy between 28.0 and 31.0) analyzer, respectively.

BMDCs were washed, lysed, and prepared with Laemmli Buffer (Boston BioProducts). Samples were run on an SDS-PAGE gel and transferred to a nitrocellulose membrane (BioRad), which was blocked in 5% BSA. Protein expression was detected with Antibodies specific for Caspase-1 (p20) from AdipoGen and GAPDH from Cell Signaling.