Mouse CD4+ T cells differentially express LPA1–6 over the course of T cell activation and polarization.

<p>(<b>A</b>). Naïve mouse CD4+ T cells were collected and activated by plate-bound anti-CD3 and anti-CD28 antibodies for 24, 48, or 72 hours. At each time point, cells were harvested and the mRNA expression of LPA1,2,3,4,5,6 was determined by semi-quantitative real-time PCR, performed in triplicate. Expression levels were normalized to mouse GAPDH using the 2∧-deltaCt method. Relative Value Units (RVU) = 2∧-deltaCt×1000. Data are mean of three independent experiments. (<b>B</b>). Protein lysates were collected at 0, 24, and 72 hours post-activation and protein expression of LPA1, LPA2, and LPA3 was measured by Western blot. GAPDH was used as a lane loading control. Data are representative of one-two experiments. (<b>C</b>). Naïve mouse CD4+ T cells were collected and activated by plate-bound anti-CD3 and anti-CD28 antibodies and cultured under Th1 (IFN-γ, anti-IL-4), Th2 (IL-4, anti-IFN-γ), or Th17 (TGF-β, IL-6, anti-IL-4, anti-IFN-γ) polarizing conditions for 72 hours. Cells were harvested and the mRNA expression of LPA2 and LPA6 was determined by semi-quantitative real-time PCR, performed in triplicate. Expression levels were normalized to mouse GAPDH and compared to naïve CD4+ T cells using the 2∧-deltadeltaCt method. Data are mean +/− SEM of three independent experiments.