For specific TGF-β (R&D Systems)–induced HK2 activity, 2 × 106 AKR-2B cells were seeded into p100 plates in 10% FBS/DMEM for 24 hours. The medium was changed to 0.1% FBS/DMEM for 24 hours after which cells were pretreated with either dimethyl sulfoxide (DMSO) (0.1%) or SB431542 for 1 hour and then stimulated with either vehicle [4 mM HCl/0.1% bovine serum albumin (BSA)] or TGF-β (10 ng/ml) for 24 hours. Cultures were lysed in modified NP40 RIPA buffer [50 mM tris-HCl (pH 7.4), 150 mM NaCl, 0.25% Na-deoxycholate, 1% IGEPAL CA-630, 1 mM EDTA, 50 mM NaF, and complete protease inhibitor cocktail], and 1 mg of total protein and 0.05 μg of HK2 antibody (Thermo Fisher Scientific) were applied to a Catch-and-Release column (Millipore) as instructed by the manufacturer. Proteins were eluted after addition of 70 μl of nondenaturing elution buffer, and HK2 activity was detected using an HK activity kit (ScienCell Research Laboratories).

For general HK activity detection, 2 × 105 AKR-2B cells were seeded into 12-well plates in 10% FBS/DMEM for 24 hours. The medium was changed to 0.1% FBS/DMEM, and cells were then treated with either DMSO (0.1%) or Lonidamine (25, 50, or 100 μM) (Bio-Techne) for 12 hours. HK activity was determined as above.

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