Gal-1 promoted TCR-agonist/MHC binding and intensified the transient negative selection ERK signaling signature. Tetramer staining decay kinetics for gal-1^−/− OT-1 DP thymocytes incubated with or without 0.1 μM rgal-1 and SIINFEKL/K^b tetramer (A,B). (A) Plot of the natural logarithm of normalized fluorescence versus time after anti-K^b addition of untreated (■) and rgal-1–treated (♦) gal-1^−/− OT-1 DP thymocytes. (B) Quantitative slope analysis of tetramer staining decay kinetics at the 0-20 minute interval between rgal-1–treated (▴, n = 6) and untreated (•, n = 6) DP populations. The slope was determined by ln (F_a/F_b)/t, where F_a is total fluorescence at the beginning of the interval and F_b is fluorescence at the end of the interval, and t is time, in hours, of interval. The horizontal bar represents the mean. To determine statistical significance, paired 2-tailed Student t tests were performed between untreated and rgal-1–treated samples. P value is listed in the panel. (C) The intensity of pERK MFI in agonist anti-CD3 antibody stimulated gal-1^−/− DP thymocytes from C57Bl/6 mice treated with (broken line) or without (solid line) 20 μM rgal-1. (D) Histograms of pERK staining in DP thymocytes from gal-1^−/− C57Bl/6 mice stimulated with anti-CD3 antibody for 2 minutes in the absence (thin line) or presence of 20 μM rgal-1 (thick line) or unstimulated (shaded). Results represent 4 independent experiments. (E) The mean fluorescent intensity (MFI) of pERK in untreated or 20 μM rgal-1–treated gal-1^−/− OT-1 DP thymocytes stimulated with agonist SIINFEKL/K^b tetramers for 2 minutes.