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. 2021 Sep 14;478(17):3331–3349. doi: 10.1042/BCJ20210425

Figure 2. TIM-3 modulates CD28 phosphorylation after αCD3/αCD28 stimulation.

Figure 2.

Changes in CD28 phosphorylation following TCR activation with αCD3/αCD28 as in Figure 1 were monitored by Western blotting using phospho-specific antibodies. (A) Representative Western blot of pY191 in CD28 for TIM-3+ and parental NF-κB reporter Jurkat cells. Grb2 was used as a loading control as described [44]. Quantification bar graphs indicate mean band intensity ± SD (n = 6 biological repeats). Two-tailed, unpaired Student's t-test was used to determine P-values. (B) Representative Western blot of pY218 in CD28 for TIM-3+ and parental NF-κB reporter cells, with quantitation below the blot. Grb2 was used as a loading control. Bars indicate mean band intensity ± SD for the 5 min time point (seven biological repeats) with P-values determined by two-tailed, unpaired Student's t-test. (C) Parental, TIM-3+, and PD-1-expressing NF-κB reporter Jurkat cells were stimulated for 5 min with αCD3/αCD28 (1 μg/ml each) with- or without the PI3K inhibitor, wortmannin (0.2 μM) to show that PTEN deficiency does not account for the effect of TIM-3 expression on CD28 phosphorylation.