Hypothalamic Gene and Protein Expression

Peripheral tissues were rapidly dissected, snap frozen, and stored at −80 °C until use. Hypothalamic blocks were micro-dissected from 300 μm coronal brain slices. RNA extraction and qPCR was performed as previously described38 (link) (qPCR primers listed in Supplementary Table S2). For protein extraction, frozen tissues were homogenised in Tissue-Protein Extraction Reagent (Peirce) containing Protease Inhibitor Cocktail (Roche) and 1 mM PMSF. Protein levels were determined by SDS-PAGE and Western blot analysis using anti-PPARγ (C26H12, Cell Signalling, 1:500) and anti-GAPDH (FL-335, Santa Cruz Biotechnology, 1:1000) antibodies.

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Cunningham P.S., Ahern S.A., Smith L.C., da Silva Santos C.S., Wager T.T, & Bechtold D.A. (2016). Targeting of the circadian clock via CK1δ/ε to improve glucose homeostasis in obesity. Scientific Reports, 6, 29983.

Publication 2016

Protease Inhibitor Cocktail anti-PPARγ (C26H12, anti-GAPDH (FL-335,

Antibodies Brain Frozen Gapdh Hypothalamic Pparγ Primers Protease inhibitor Protein Sds page Tissue Western blot analysis

Corresponding Organization :

Other organizations : University of Manchester, Pfizer (United States)

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Variable analysis

independent variables

Peripheral tissue dissection
Hypothalamic block micro-dissection

dependent variables

RNA expression (qPCR)
Protein levels (Western blot)

control variables

Tissue storage temperature (-80 °C)
Tissue homogenization buffer (Tissue-Protein Extraction Reagent with Protease Inhibitor Cocktail and PMSF)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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