Lightcycler 480 rt pcr

DSF experiments were performed as described^{8 (link), 13 (link)} using 5 µM protein, 2.5 µM DNA when indicated and various concentrations of compound (BAD, carba-NAD⁺, benzamide, and ADP-ribose) as indicated in Fig. 1 and Fig. 3. Experiments were performed on a Roche LightCycler 480 RT-PCR. Reactions including DNA were performed with a 26- or 28-base pair (bp) unphosphorylated oligonucleotide for PARP-1, a 28-bp 5´-phosphorylated (5´P) oligonucleotide for PARP-2, and a 47-bp oligonucleotide including a central 5´P nick for PARP-3¹³ in the following buffer: 25 mM Hepes pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.1 mM TCEP. Reactions with PARP-3 were conducted at lower ionic strength to allow binding to DNA as described (25 mM Hepes pH 8.0, 50 mM NaCl, 1 mM EDTA and 0.1 mM TCEP)^{13 (link)}. ΔT_M values were calculated by subtracting the T_M determined for the protein in the absence of compound from the T_M determined in the presence of compound. Experiments were performed in triplicate and a Boltzmann sigmoid was fit to the data to determine the T_M values (KaleidaGraph).

The reaction cDNA was reverse transcribed using Prime-Script^TM RT Master Mix (#RR036B, TAKARA, Japan) and FastStart Universal SYBR Green (#4913850001, Takara, Japan) was used for the qRT-PCR on a Lightcycler480 RT-PCR instrument (Roche
Diagnostics, Penzberg, Germany). GAPDH was used as a standardized internal control. The quantification was performed with the 2^{−△△CT of each sample}/2^{−△△CT of the control} method and primers in this study were shown in Table 1.Table 1.

Primers for qRT-PCR in this study

Names	Sense (5’−3’)	Antisense (5’−3’)
GBP2b	GAGTACTCTCTGGAA	TAGATGAAGGTGCTG
AMPK	AAAGGGTACACAGACGCCAG	CTCCGAATCTTCTGCCGGTT
ULK1	CCACTTGGGGAGAAGGTGTG	ACTCAACAGCAGACAGCCAG
mTOR	CCGCTACTGTGTCTTGGCAT	CAGCTCGCGGATCTCAAAGA
Egr3	GCCTGACAATCTGTACCCCG	TCCATCACATTCTCTGTAGCCA
GBP5	GAACGCCAAAGAAACAGTGAG	GAATAGCCTCCAACCTCTGTG
Itgb8	ACTGGGCCAAAGTGAACACA	TCTTGAACACACCATCCGCA
Ptgs2	CATCCCCTTCCTGCGAAGTT	GGCCCTGGTGTAGTAGGAGA
Socs1	AGCAGAGAGAACTGCGGC	GCTGGCGGCAGGACG
Ptgs2	CATCCCCTTCCTGCGAAGTT	GGCCCTGGTGTAGTAGGAGA
Cxcl 5	CACTCGCAGTGGAAAGAACG	CGTGGGTGGAGAGAATCAGC
Cxcl 3	GAAAGGAGGAAGCCCCTCAC	ACACATCCAGACACCGTTGG
Cxcl 2	GCTGTCCCTCAACGGAAGAA	CAGGTACGATCCAGGCTTCC
IL-1β	TGCCACCTTTTGACAGTGATG	TGATGTGCTGCTGCGAGATT

Differential scanning fluorimetry experiments were performed essentially as described
(20 (link)) using 5 μM protein and 2.5 μM DNA when
indicated. Fluorescence emission was measured as the temperature was increased from 20 to
85°C on a Roche LightCycler 480 RT-PCR. In Figure 5C, the melting temperature in the absence of DNA was subtracted from the
T_M in the presence of a 28-bp duplex 5′P (PARP-2) or a
47-bp duplex with 5′P nick (PARP-3). Reactions for PARP-3 were conducted at lower
ionic strength (25 mM HEPES pH 8.0, 50 mM NaCl, 1 mM EDTA and 0.1 mM TCEP).
The ΔT_M values shown represent an experiment
performed in triplicate. A Boltzmann sigmoid was fit to the data to determine
T_M values (KaleidaGraph).