Supplementary Table 1
Comaparison of EGFR levels in rip1+/+/tnfr1+/+ MEFs and rip1+/+/tnfr1-/- MEFs by
real-time PCR:
RNA was extracted from rip1+/+/tnfr1+/+ MEFs and rip1+/+/tnfr1-/- MEFs by RNeasy
Kit (Qiagen),reverse transcribed to cDNA using random primers (Invitrogen) and
superscript II reverse transcriptase (Invitrogen) and quantified using real-time PCR (ABI
Prism 7700, Applied Biosystems, CA). Ct values were obtained using the Sequence
Detector 1.1 software. The fold difference between the two cell lines is calculated as
R = 2-(delta ct cell line1-delta ct cell line2) where R is the relative expression of the gene of interest,
ct is the cycle threshold, and delta ct is the difference in the ct value of the cell lines when
compared with GAPDH after normalization. Each reaction was run in triplicate in the
presence of SYBR-GREEN (Applied Biosystems) and repeated at least two times
independently.
EGFR primers used for Real Time PCR:
Forward primer: AGG CAC AAG TAA CAG GCT CAC
Reverse primer: AAG GTC GTA ATT CCT TTG CAC
Cell line
Mean
SEM
Normalised ct
value for EGFR
rip1+/+/tnfr1+/+ 22.64
3.6
MEFs
rip1+/+/tnfr1-/37.30
0.17
MEFs
pvalue
0.03
Results:
The difference in EGFR mRNA transcript between the two cell lines was found to be 215.
Thus, there is significantly more EGFR mRNA in rip1+/+/tnfr1+/+ MEFs compared to
rip1+/+/tnfr1-/- MEFs.