Bullet blender bead lysis kit

Protein from frozen mouse hearts were prepared by lysis in ice-cold RIPA buffer (ThermoFisher, cat.89900) containing protease and phosphatase inhibitor (ThermoFisher, cat. A32959). Tissue was homogenized using bullet blender bead lysis kit (Next Advance), and protein concentrations were determined with Pierce BCA Protein Assay Kit (Thermofisher, cat. 23225). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4–15% gradient gel (Bio-Rad, cat.4561086) and transferred to PVDF membrane using iBlot 2 transfer system (ThermoFisher). Protein expression was measured by chemiluminescence using ChemiDoc imaging system (Bio-Rad). Proteins were detected with the following primary antibodies: BCKDH (ThermoFisher, cat.PA5-97248), phospho-BCKDH (S293) (abcam, ab200577), PP2Cm (abcam, ab135286), 4-hydroxynonenal (abcam, ab46545) (14 (link), 56 (link), 57 (link)).

Total RNA was extracted from cell cultures using the EZ-RNA Total RNA Isolation Kit (Biological Industries) and used in quantitative real-time PCR (qRT-PCR) as previously described [18 (link)]. From harvested organs, total RNA was extracted using the Bullet blender bead lysis kit (Next advance Inc., Averill Park, NY, USA) and the EZ-RNA Total RNA Isolation Kit (Biological Industries). Detection of human neuroblastoma cells (micro-metastases) in mouse tissue was performed as previously described [18 (link)], using specific primers for the human/mouse β2 microglobulin. Primers used for mRNA amplification were: human β2 microglobulin (NM_004048) For-5′-ATGTAAGCAGCATCATGGAG-3′, Rev-5′-AAGCAAGCAGAATTTGGAAT-3′; mouse β2 microglobulin (NM_009735.3) For-5′-CTGGTCTTTCTGGTGCTTGT-3′, Rev- 5′-GGCGTGAGTATACTTGAATTTGAG-3′; PHOX2B (NM_003924.3) For-5′-TACGCCGCAGTTCCTTACAA-3′, Rev-5′-GAAGACCCTTTCCAGCTCTTT-3′; TH (NM_000360.3) For-5′-TGTACTGGTTCACGGTGGAGTT-3′, Rev-5′-AATCTCAGGCTCCTCAGACA-3′; GATA3 (NM_001002295.1) For-5′-ACACTCTGGAGGAGGAATGC-3′, Rev-5′-CTGGATGCCTTCCTTCTTCATA-3′; RPS13 (NM_001017.2) For-5′-CGAAAGCATCTTGAGAGGAACA-3′, Rev-5′-TCGAGCCAAACGGTGAATC-3′.

Frozen liver biopsy specimens were minced and lysed using the Bullet Blender Bead Lysis kit (Next Advance; Troy, NY). Liver genomic DNA was extracted with the ALLprep DNA/RNA Mini Kit (Qiagen; Valencia, CA) and quantified using the Qubit system (ThermoFisher Scientific; Waltham, MA). Whole genome DNAm profiling was performed using the Infinium MethylationEPIC BeadChip kit (Illumina; San Diego, CA). We used the EZ DNA Methylation Kit (Zymo Research; Irvine, CA) for sodium bisulfite conversion of DNA, and bisulfite-treated DNA was fragmented and hybridized to the bead chips. Bead chips were scanned and imaged with the iScan system (Illumina). DNAm levels for each CpG residue were estimated as the ratio of the methylated signal intensity over the sum of the methylated and unmethylated intensities at each locus.

Flash-frozen tumor samples were lysed using Bullet Blender Bead Lysis Kit (Next Advance, Pink5E100, Troy, NY, USA). PDGF-AA levels from in vivo tumor lysates were analyzed by ELISA using the Human PDGF-AA ELISA kit (Thermo; EHPDGFA) according to manufacturer’s protocol. Samples were assayed in triplicate, and results represent average mean concentration over triplicate experiments.