|
|
Blood, Vol. 109, Issue 8, 3139-3146, April 15, 2007
Serotonin provides an accessory signal to enhance T-cell activation by signaling through the 5-HT7 receptor
Blood León-Ponte, et al.
109: 3139
Supplemental materials for: León-Ponte et al, Vol 109, Issue 8, 3139-3146
RT-PCR for DAT and NET Expression of DAT, NET, and SERT was analyzed by PCR using cDNA equivalent to 50 ng of total RNA. For NET, SERT, and GAPDH, first-strand cDNA synthesis was performed using Advantage RT-for-PCR (BD Biosciences) using an oligo (dT)18 primer. Primers for SERT and GAPDH were reported previously.1 NET primers were as follows: forward, 5′-CCA TAC CAA ATA CTC CAA ATA CAA GTT-3′, and reverse, 5′-CGT CAA GAG TTT CCG GTG TCG CTT-3′. For DAT, first-strand cDNA synthesis and subsequent amplification was performed using the One Step RT-PCR (Qiagen, Valencia, CA) using DAT-specific primers (forward, 5′-ACT GGA CGC TTA CAG GAC CTC AAA G-3′, and reverse, 5′-CTC CCG CTC TTG AAC CTC CAC TGG T-3′). All transcripts were amplified for 36 cycles from cDNA equivalent to 50 ng total RNA. Annealing temperatures were as follows: NET (54°C), DAT (53°C), SERT (58°C), and GAPDH (57°C). PCR products were resolved as single bands by agarose gel electrophoresis and visualized with SYBR Safe (Molecular Probes) fluorescence.
Files in this Data Supplement:
- Figure S1. Relative expression of 5-HT1B and 5-HT7 receptors by T cells (JPG, 44.9 KB)
-
T cells were negatively purified, activated with Con A or PMA plus ionomycin. Gene expression for 5-HT1B or 5-HT7 receptors was examined by RT-PCR using cDNA equivalent to 50 ng total RNA and 32 cycles of amplification. Primers for the 5-HT1B receptor and the type 7 5-HTR family amplify a product encoded within 1 exon; thus, both cDNA- and RT-negative (RNA) samples are shown. Comparable quantities of cDNA were ensured by amplification of GAPDH.
- Figure S2. Expression of monoamine transporters by T cells (JPG, 32.2 KB)
-
T cells were negatively purified and activated with Con A or PMA plus ionomycin, and gene expression for SERT, NET, and DAT was examined by RT-PCR. Comparable quantities of cDNA were ensured by amplification of GAPDH.
REFERENCE
1. O’Connell PJ, Wang X, Leon-Ponte M, Griffiths C, Pingle SC, Ahern GP. A novel form of immune signaling revealed by transmission of the inflammatory mediator serotonin between dendritic cells and T cells. Blood. 2006;107:1010-1017.
| |
