Figure 8.
preTCR expression can drive tonic signaling in a T cell line. (A) Expression of the αβTCR (anti-αβTCR Ab) and preTCR (mScarlet) complexes on Jurkat T cells (“TCR positive”), Jurkats subjected to CRISPR/Cas9-mediated disruption of the TCRA gene (“TCR negative”) and these cells transduced with pTa-mScarlet to drive preTCR expression (preTCR). Boxes denote the color used throughout the figure for each cell line. (B) Surface staining for CD3 ε was used to confirm preTCR expression. TCR+ve Jurkat cells (blue) show intense CD3ε staining that is completely lost on TCR−ve cells (green) when compared to isotype control (filled grey). Expression of pTa-mScarlet (red) drives CD3ε surface staining demonstrating preTCR complex formation in these cells. (C) Flow cytometry plots showing all three cell lines could be easily separated when mixed in one experiment. (D) Left panel shows representative flow data for CD69 expression on the three Jurkat variants, colored as in B, and the right panel shows the quantification of CD69 expression. Error bars show mean ± SEM (n = 6); asterisks indicate P < 0.05 (*) or P < 0.01 (**) when comparing indicated datasets. (E) Equivalent datasets to D for CD6 expression. (F) Equivalent datasets to D for CD5 expression. (G) Representative Western images when blotting for denoted (phospho-)proteins for lysed samples of the three Jurkat variants shown in A. Relative total protein normalization (TPN) values under each blot correct for sample loading differences. (H) Quantification of Western images as shown in G, where each replicate is shown with connecting lines. Integrated band intensities were corrected for loading using TPN values. Bars show mean (n = 3). A two-tailed, two-sided t test was used for all statistical analyses. Source data are available for this figure: SourceData F8.

preTCR expression can drive tonic signaling in a T cell line. (A) Expression of the αβTCR (anti-αβTCR Ab) and preTCR (mScarlet) complexes on Jurkat T cells (“TCR positive”), Jurkats subjected to CRISPR/Cas9-mediated disruption of the TCRA gene (“TCR negative”) and these cells transduced with pTa-mScarlet to drive preTCR expression (preTCR). Boxes denote the color used throughout the figure for each cell line. (B) Surface staining for CD3 ε was used to confirm preTCR expression. TCR+ve Jurkat cells (blue) show intense CD3ε staining that is completely lost on TCR−ve cells (green) when compared to isotype control (filled grey). Expression of pTa-mScarlet (red) drives CD3ε surface staining demonstrating preTCR complex formation in these cells. (C) Flow cytometry plots showing all three cell lines could be easily separated when mixed in one experiment. (D) Left panel shows representative flow data for CD69 expression on the three Jurkat variants, colored as in B, and the right panel shows the quantification of CD69 expression. Error bars show mean ± SEM (n = 6); asterisks indicate P < 0.05 (*) or P < 0.01 (**) when comparing indicated datasets. (E) Equivalent datasets to D for CD6 expression. (F) Equivalent datasets to D for CD5 expression. (G) Representative Western images when blotting for denoted (phospho-)proteins for lysed samples of the three Jurkat variants shown in A. Relative total protein normalization (TPN) values under each blot correct for sample loading differences. (H) Quantification of Western images as shown in G, where each replicate is shown with connecting lines. Integrated band intensities were corrected for loading using TPN values. Bars show mean (n = 3). A two-tailed, two-sided t test was used for all statistical analyses. Source data are available for this figure: SourceData F8.

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