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2.5. Phenotyping of LepRbPdynKO and control mice LepRbPdynKO and control littermates were weaned into individual housing at 21 days and fed either chow (Purina Lab Diet 5001) or high fat diet (Research Diets ""type"":""entrez-nucleotide"",""attrs"":""text"":""D12492"",""term_id"":""220376"",""term_text"":""D12492""D12492, 60% kcal from fat). Body weight was monitored weekly. A fasted (24?h) blood glucose sample was taken at 12�C14 weeks of age. Analysis of body fat and lean mass was performed between 12 and 14 weeks of age using an NMR-based analyzer (Minispec LF90II, Bruker Optics). We also analyzed a subset of mice (13�C16 weeks old) for oxygen consumption (VO2), food intake, and locomotor Liothyronine Sodium activity using the Comprehensive Laboratory Animal Monitoring System (CLAMS, Columbus Instruments). Insulin was assessed using a double-antibody radioimmunoassay using an 125I-Human insulin tracer (EMD Millipore), a rat insulin standard (Novo), a guinea pig anti-rat insulin first antibody (EMD Millipore), and a sheep anti-guinea pig gamma globulin-PEG selleck chemicals second antibody (MDRTC). Leptin was assayed by commercial ELISA (EMD Millipore). No significant differences were detected between the control (Pdyncre/+Lepr+/+, and Leprflox/flox) groups at the conclusion of the study and thus the data from these groups was combined for subsequent analysis. 2.6. Statistics Physiological data are reported as mean?��?SEM. Statistical analysis of physiological data was performed using Prism (version 6.0) software. Unpaired t-tests were used to compare results between groups of two. Body weight gain between genotypes was analyzed by two-way ANOVA. p?Lapatinib Figure?1 LepRbeGFP-L10a mice for TRAP-seq of LepRb neurons. (A) Leprcre mediates the excision of the transcription-blocking cassette from Rosa26eGFP-L10a in LepRb cells, promoting the expression of eGFP-L10a in these cells. (B,E) Representative images of GFP-IR ... 3.2. TRAP-seq analysis of hypothalamic and brainstem LepRb neurons We performed anti-eGFP TRAP on hypothalamic extracts from LepRbeGFP-L10a mice and utilized the resultant mRNA (as well as TRAP-depleted supernatant RNA) to generate multiplexed libraries for sequencing on the Illumina HiSeq2000 platform.