Fig 1. GXMGal effect on Treg cell response.

Activated PBMC (A and C) or purified CD4⁺ T cells (B) (both 5×10⁶/ml) from Control and RA were incubated for 2, 18 and 72 h in the presence or absence (NS) of GXMGal (10 μg/ml) or MTX (10 ng/ml). After 2 and 18 h (A) or 18 h (B) of incubation, cell lysates were analyzed by western blotting. Membranes were incubated with Ab to FOXP3. Actin was used as an internal loading control. Normalization was shown as mean ± SEM of five independent experiments (A and B). *, p<0.05 (triplicate samples of 5 different Control and RA; RA treated vs untreated cells). Note that for both Control and RA cells the immunoblots at 18 h (A) share Actin loading control panels with the experiment shown in Fig 6A, as the same immunoblots were stripped and re-probed using different antibodies. Culture supernatants were collected after 2, 18 and 72 h to test TGF-β1 and IL-10 levels by specific ELISA assays. *, p<0.05 (triplicate samples of 7 different Control and RA; RA GXMGal-treated vs untreated cells); †, p<0.05 (triplicate samples of 7 different Control and RA; RA MTX-treated vs untreated cells) (C).