Enolase

In vitro auto- and trans-phosphorylation assays were preformed as described earlier9 (link). Cos-7 cells were transfected with cDNA encoding the EphA2 construct in question (endogenous EphA2 was precipitated from MDA-MB-231). Cells were lysed 18 h later and EphA2 was IP. Following IP, beads were washed three times with HNTG buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 100 μM sodium vanadate). Samples were dephosphorylated using calf intestinal phosphatase (#M0290, 0.5 U μg⁻¹ protein) for 60 min at RT and then washed twice with kinase reaction buffer KRB (25 mM HEPES (pH 7.5), 2.5 mM MgCl₂, 4 mM MnCl₂, 100 μM sodium vanadate). Beads were resuspended in KRB containing 5 μg of acid denatured enolase (Sigma #E0379) at RT for the indicated time, before stopping the reaction by adding SDS/PAGE sample buffer and heating to 100 °C. Autophosphorylation of Eph or transphosphorylation of enolase was detected using PY72.

LC-MS grade solvents (water,
acetonitrile, and isopropanol), formic acid (FA), and trifluoroacetic
acid (TFA) were purchased from Thermo Fisher Scientific (Waltham,
MA) or Merck (Darmstadt, Germany). Trypsin and trypsin/Lys-C proteases
(MS grade) were from Promega (Madison, WI); RapiGest detergent was
from Waters (Eschborn, Germany), and other common chemicals were from
Sigma-Aldrich (Munich, Germany). Polyacrylamide gradient gels (4–20%)
were from Serva Electrophoresis GmbH (Heidelberg, Germany). Protein
standards, glycogen phosphorylase (GP), alcohol dehydrogenase (ADH),
enolase (ENO), and ubiquitin (UBI), were purchased as a lyophilized
powder from Sigma-Aldrich. The reference protein standard (BSA, Pierce
grade, in ampules) was from Thermo Fisher Scientific (Waltham, MA).
Isotopically labeled amino acids (¹³C₆,¹⁵N₄-l-arginine (R) and ¹³C₆-l-lysine (K)) were purchased from Silantes GmbH
(Munich, Germany).

Six branched dextrans in the mass range of 23.8 to 667.8 kDa and following proteins of different sizes and amounts of glycosylation (refer to Supporting Information Table 1) were purchased from Sigma‐Aldrich (Steinheim, Germany): β‐galactosidase (E. coli), enolase (baker's yeast), carbonic anhydrase (from bovine erythrocytes), immunoglobulin G (IgG, bovine), ovalbumin (chicken) and transferrin (human). For calibration with linear polysaccharides, five pullulan standards in the mass range of 22.8 to 788 kDa (provided by S. Alban, Kiel University, Kiel, Germany) and six OBGs in the mass range of 31–1508 kDa (Putus Macromolecular Science & Technology, Wuhan, Hubei, China) were used. Ammonium acetate (≥99.99%) and ammonium hydroxide (ACS reagent) were purchased from Sigma Aldrich (Steinheim, Germany).

Recombinant PGAM1 enzymatic activity was measured by a multiple enzymes coupled assay. In brief, assay was conducted in 10 μL buffer (50 mM HEPES, PH 7.5, 10 mM MgCl₂) containing 5 μL 1 mg/ml recombinant PGAM1 enzyme, 2.5 μL test compound, 2.5 μL 1 mM ADP, 1 mM 3-PG, 0.5 units/ml enolase (Sigma–Aldrich) and 0.5 units/mL recombinant pyruvate kinase M1 (Sigma–Aldrich). The production of ATP was measured by Kinase-Glo^® Max Luminescent Kinase Assay (Promega).

We used the following antibodies: polyclonal anti-STEP, anti-Pyk2 (pY402), anti-ERK1/2 (pT202/pY204), and anti-ERK1/2 from Cell Signaling Technology (Danvers, MA, USA); polyclonal anti-Pyk2, monoclonal anti-β-actin, and polyclonal anti-fyn from Santa Cruz Biotechnology (Santa Cruz, CA, USA); monoclonal anti-v-src (Ab1, clone 327) from Calbiochem (EMD Chemical, Merck, Darmstadt, Germany); polyclonal anti-GluN2B (pY1472), anti-GluN2B, and monoclonal anti-phosphotyrosine (pY, clone 4G10) from Millipore Bioscience Research Reagent (Billerica, MA, USA); and peroxidase-conjugated goat anti-mouse and goat anti-rabbit from Bio-Rad (Hercules, CA, USA). Protein A/G PLUS agarose was from Santa Cruz Biotechnology and Trysacryl-immobilized protein A from Thermo Scientific (Waltham, MA, USA). Nitrocellulose was from Schleicher and Schuell Bioscience Inc. (Dassel, Germany); p-nitrophenyl phosphate (p-NPP) and enolase were from Sigma Chemical (St. Louis, MO, USA). Complete protease inhibitor cocktail was from Roche Diagnostics (Basel, Switzerland). [γ³²P] ATP (>3000 Ci/mmol) was obtained from DuPont NEN (Boston, MA, USA).