Comprehensive Gene Expression Analysis

RNA isolation, cDNA synthesis and qPCR were performed at day 8 of differentiation (or as stated in the figure legends) as previously described [6] (link). Control reactions for contaminating DNA were performed routinely and relative expression calculated using the Pfaffl method [29] (link). The geometric mean of three stable reference genes (Nono, Ywhaz and Actb or as stated in the figure legend) were obtained from five commonly used sequences and used for normalisation. Primer sequences available on request, some of which were obtained from PrimerBank [30] (link).
SDS-PAGE was performed and transferred to nitrocellulose membranes as described previously [31] (link). Antibodies against p-eIF2α (#9721), eIF2 α (#5324S), p-p38 MAPK (#9211), p38 MAPK (#8690S), Beclin1 (#3495), LC3B (#3868), p-Akt Ser473 (#9271) were from Cell Signalling, SH-PTP2 (sc-280) and Akt1/2/3 (sc-8312) from Santa Cruz. All antibodies were detected with goat anti-rabbit HRP secondary antibody (#28177) from Anaspec. Proteins were visualized using enhanced chemiluminescence (ECL) and quantified by densitometry scanning using the Fusion imaging system and Bio-1D software (Peqlab).

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Mcilroy G.D., Tammireddy S.R., Maskrey B.H., Grant L., Doherty M.K., Watson D.G., Delibegović M., Whitfield P.D, & Mody N. (2016). Fenretinide mediated retinoic acid receptor signalling and inhibition of ceramide biosynthesis regulates adipogenesis, lipid accumulation, mitochondrial function and nutrient stress signalling in adipocytes and adipose tissue. Biochemical Pharmacology, 100, 86-97.

Publication 2016

p-Akt Ser473 Akt1/2/3 (sc-8312) Bio-1D software

Akt1 Anti antibody Antibodies Beclin1 Cdna Chemiluminescence Eif2 Genes Goat Isolation Membranes Nitrocellulose P38 mapk Primer Proteins Rabbit Sds page Sh ptp2 Synthesis

Corresponding Organization : University of Aberdeen

Other organizations : University of the Highlands and Islands, University of Strathclyde

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Variable analysis

independent variables

Not explicitly mentioned

dependent variables

Gene expression levels measured by qPCR
Protein levels measured by Western blot

control variables

Time point of differentiation (day 8 or as stated)
Reference genes used for normalization of qPCR data (Nono, Ywhaz, Actb or as stated)
Procedures for RNA isolation, cDNA synthesis, and qPCR as previously described [6]
Procedures for SDS-PAGE and Western blotting as previously described [31]
Antibodies used for Western blot analysis

controls

Control reactions for contaminating DNA were performed routinely
Relative expression was calculated using the Pfaffl method [29]

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