Figure 2
Expression of SH2D2A variants in primary CD4+ T cells. A. Expression of SH2D2A transcript variants in anti-CD3 stimulated CD4+ T cells: mRNA expression levels of SH2D2A variants relative to Zap-70 were assessed by RT-PCR in CD4+ T cells stimulated with CD3 ligation for 2 hours from four healthy blood donors. Results shown are median values ± SD of the estimated level of alternative SH2D2A transcripts relative to the total amount of SH2D2A transcripts. The SH2D2A-4 variant was not always observed. The median value of the SH2D2A-4 is 0,03% ± 0,2%. B: The relationship between the different SH2D2A transcripts does not vary significantly throughout anti-CD3 stimulation of CD4+ T cells: Primary CD4+ T cells were stimulated with anti-CD3 for 24 hours, and total amount of SH2D2A transcript (1–5) and mRNA levels of SH2D2A transcript variants 2, 3, 4 or 5 were assessed at different time points. After the initial two hours of anti-CD3 stimulation, the relationship between the five SH2D2A transcripts remained constant throughout the stimulation period. Results shown are median values of TSAd transcripts relative to Zap-70 transcripts. C: TSAd of 37 kDa is expressed in CD3 stimulated primary CD4+ T cells. Primary CD4+ T cells were stimulated with anti-CD3 for 2 and 4 hours and total sonicated cell lysates were separated by SDS-PAGE and immunoblotted with TSAd Abs and Zap-70 mAbs as a loading control. TSAd Abs detects two CD3 induced bands of 52 kDa and 37 kDa respectively. D. Expression of TSAd of 52 and 37 kDa is repressed by siRNA treatment of CD4+ T cells: Primary CD4+ T cells were transfected with control medium (0) or increasing concentration of TSAd siRNA (0,05, 0,5 or 5 μM p690) or with 5 μM control siRNA (C = TSAd p369) using Amaxa electroporator and stimulated with anti-CD3 beads for 24 hours. Total sonicated cell lysates were separated by SDS-PAGE and immunoblotted with TSAd Abs and Zap-70 mAbs as a loading control (not shown). Lysates of Jurkat TAg cells stably transfected with HA-tagged TSAd cDNA (3a3) was included as a blotting control. TSAd Abs detects two bands of 52 kDa and 37 kDa that can be inhibited by siRNA, and one non-specific band of 45 kDa, which is not affected by siRNA treatment.