Fig 1: SFRP2 increases CD38 and PD-1 mRNA and protein levels in T-cells. (A) Splenic T-cells cultured in IL2-containing medium were treated with or without SFRP2 (30 nM) for 1 h, and the mRNA levels for CD38 and PD-1 were measured by qRT-PCR (n = 8). GAPDH was used as an internal control. (B–F) All T-cells were treated with IL-2. Samples were probed using Western blot with antibodies to the indicated protein markers. Actin was the loading control for cytoplasmic fraction; histone H3 and TATA were loading controls for nuclear fractions. (B,C,E,F) Int. ratio: intensity ratios listed below Western blots for (B–F) were calculated after normalization to both loading control and reference samples (indicated by 1.00). (B,C) Cytoplasmic fractions of wild-type splenic T-cells were isolated and protein levels of CD38 (B) and PD-1 (C) were increased in SFRP2-treated T-cells, compared to untreated cells. (D) Western blot probing for FZD5 protein in mouse splenic T-cells. % actin: total band intensity was normalized to actin. (E) Splenic T-cells were untreated or treated with recombinant SFRP2 protein for 1 h. The nuclear fraction was isolated from the T-cells, and nuclear NFATc3 and ß-catenin protein levels were measured. Nuclear fractions demonstrated increased NFATc3 protein levels but no change in ß-catenin levels with SFRP2 treatment. (F) Splenic T-cells were activated with antigen gp100 (0.87 µM) with or without 10 µM of hSFRP2 mAb treatment. Nuclear fractions demonstrated decreased NFATc3 protein levels in hSFRP2 mAb-treated cells activated by antigen.