Rabbit anti hexokinase

Western blotting was performed using a standard TCA-based protocol. Briefly, 2.5 OD units of culture were treated with 5% TCA at 4C for at least 10 min. Samples were then washed with 1 mL acetone. Acetone was aspirated and pellets were dried overnight at RT. Lysates were made by adding 100 mL protein lysis buffer [50 mM TE, 3 mM DTT, 1.1 mM PMSF (Sigma), 1 μM pepstatin A, 1X protease inhibitor cocktail (Roche)] and 1 volume acid-washed glass beads (Sigma), and bead-beating for 5 min at RT. 3X SDS loading buffer was added and samples were boiled for 5 min. Beads were pelleted by centrifugation and 5 mL supernatant was loaded onto 4–12% Bis-Tris polyacrylamide gels. Following electrophoresis, proteins were transferred using a semi-dry transfer apparatus (Trans-Blot Turbo, BioRad). The following antibodies were used: mouse anti-V5 (Invitrogen, 1:2,000), rabbit anti-hexokinase (Stratech, 1:10,000), anti-mouse and anti-rabbit secondaries (Li-Cor, 1:15,000). Primary antibody incubation was overnight, secondary for 1–2 hr. Blots were visualized and quantified using a Li-Cor system.