Anti hk2 antibody

Total proteins were isolated from CC cells, which were lysed on ice with a RIPA lysis buffer (Cell Signaling Technology, MA). The protein concentration was determined using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Extracted protein (30 µg) was separated by 12% SDS-PAGE and transferred onto a PVDF membrane (GE Healthcare Life Sciences, Piscataway, NJ). The membranes were blocked with 5% non-fat milk for 1 h and then incubated with anti-HK2 antibody (1:1000, #2106, Cell Signaling) and anti-GAPDH antibody (1:5000, #2118, Cell Signaling) at 4°C overnight. Then, the membranes were subsequently incubated with secondary antibodies for 2 h at room temperature. The protein bands were detected using an ECL detection kit (Amersham Pharmacia Biotech, UK).

Samples of 144 human breast cancer and 114 normal tissues were obtained from the PLA General Hospital, with the informed consent of patients and approval of the Institutional Review Committees of the Chinese PLA General Hospital. The expression level of let-7b-5p was determined following miRNA FISH instructions (Exonbio). Let-7b-5p probe (FITC labeled) sequence was AACCACACAACCTACTACCTCA. The scramble probe (negative control) sequence was GTGTAACACGTCTATACGCCCA. The level of HK2 expression was determined by IHC and cyanine 3 system (K1051, APExBIO). Anti-HK2 antibody (Cell Signaling Technology) was used as the primary antibody. IHC of specimens was analyzed as previously described [49 (link)]. The fluorescence intensity was examined using a microscope (BX53F; Olympus, Tokyo, Japan). The let-7b-5p or HK2 score was calculated by multiplying staining intensity (1, low; 2, medium; 3, strong) by stained cells percentage (0–100%).

Let-7b-5p mimic/inhibitor was purchased from GenePharma. Wild-type and mutated sequences of the HK2 3′-UTR were inserted into a pcDNA3-luciferase expression vector, generating HK2 3′-UTR WT and HK2 3′-UTR MUT, respectively. HK2 expression vector was constructed by inserting PCR-amplified fragments into pcDNA3 (Invitrogen). HK2 shRNA stable cell line was established by lentiviral transduction using pSIH-H1-Puro (System Biosciences) carrying HK2 shRNA. The target sequence of HK2 shRNA was ATAAGCTACAAATCAAAGA. Stable cells that were infected with lentiviruses were screened using puromycin. Reagents for miRNAs and plasmids transfection were, respectively, Lipofectamine RNAiMAX and Lipofectamine 3000 (Invitrogen). Anti-HK2 antibody was obtained from Cell Signaling Technology and an anti-β-actin antibody was obtained from Santa Cruz Biotechnology.