SUPPLEMENTARY DATA
Subcutaneous adipose tissue RT PCR. Master mix consisted of 4 µl 5x miScript RT Puffer, 1 µl
RNase Inhibitor 10 U/µl (Invitrogen, Carlsbad, USA) and 1 µl miScript Reverse Transcriptase Mix per
sample. The total volume of 20 µl contained 500 ng RNA in RNase-free water and was incubated for 60
min. at 37°C and for 5 min. at 95°C. Master Mix consisted of 12.5 µl 2x QuantiTect SYBR Green PCR
Master Mix and 2.5 µl QuantiTect Primer per sample and was added to 10 µl cDNA diluted in RNasefree water. For detecting expression levels of the target genes, we used 10 ng cDNA for IL-6, 25 ng
cDNA for TNFα and 5 ng cDNA for TLR2 and TLR4 as well as 0.05 ng cDNA for the reference gene
PPIA. The RT-PCR started with an initial activation step of 95°C for 15 min. The 3-step cycling
consisted of denaturation (94°C for 15 s), annealing (55°C for 30 s) and extension (72°C for 30 s) and
was repeated 39times. Finally we performed a melting curve analysis and incubated the samples at 95°C
for 15 s, at 50°C for 20 s with a final increase by 0.3°C until reaching 95°C for 15 s.
Supplementary Figure. Time courses of blood glucose levels (A) and glucose tracer enrichment (B).
iv fat (red), po fat (blue), LPS (green), control (black). Tracer enrichment is shown as incremental
tracer-to-tracee ratio.
©2013 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1179/-/DC1
SUPPLEMENTARY DATA
©2013 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1179/-/DC1