Rabbit anti hk2 antibody

Binding assay is conducted on Pierce Nickel-coated 96-well plates and after successive incubation with His-PRKAR1a and Myc-HK2 as described above, unbound proteins will be washed and blocking buffer will be added for 1 h at room temperature. Thereafter, GST-GSK3β is added at increasing concentration (0–64 nM; 40 μl/well, 2 h, room temperature). For G6P-induced binding inhibition, 30 min before the end of the incubation, G6P (0–1000 μM) is added directly to the wells. Unbound protein was removed by washing the wells as described above and bound GST-GSK3β was detected with monoclonal anti-GST HRP-conjugated antibody (Abcam; 1:5000 in blocking buffer, 1 h, room temperature). Bound PRKAR1a or HK2 were detected by incubation with rabbit anti- PRKAR1a antibody (Cell Signaling; 1:2000 in blocking buffer) or rabbit anti-HK2 antibody (Cell Signaling; 1:2000 in blocking buffer) and HRP-conjugated anti-rabbit IgG (1:3000, 1 h, room temperature). For each assay, non-specific binding is also conducted as described in figures and HRP reaction is conducted as described above.

Human oral tumor IHC was performed according to previous descriptions [16 (link)]. Briefly, sections of miR-143 low or high expression were deparaffinized in xylene. Then, samples were rehydrated through a gradient concentration of alcohol followed by incubation by 5% normal goat serum to block non-specific staining. Sections were then incubated with rabbit anti-HK2 antibody (1:200, Cellsignaling, #2867, Danvers, MA, U.S.A.) overnight at 4°C. The slides were incubated with biotin-labeled goat anti-rabbit IgG and further incubated with streptavidin peroxidase solution (SABC kit, Boster Biological Technology, Ltd., Wuhan, China). The staining was visualized by reaction with 3,3′-di-aminobenzidine (Boster Biological Technology, Ltd.) in PBS for 5 min at room temperature. All of the IHC staining results were reviewed independently by two pathologists.