Figure 4. Increased TCR signaling and UDP-GlcNAc levels are neither necessary nor sufficient for poly-LacNAc induction.

(A) WT, TCRβ^-/- and Lck^-/- Jurkat cells were treated with SW for the indicated times and analyzed for lectin binding by flow cytometry. (B) Jurkat cells were treated as indicated for 24 hr and analyzed for LEA binding by flow cytometry. (C) Lysates of Jurkat T cells treated with SW as indicated were immune-blotted with anti-B3GNT2 and actin. (D) B3GNT enzyme activity was measured in lysates of Jurkat T cells treated with and without SW for 72 hr. (E–G) Total cellular UDP-GlcNAc levels were measured in mouse T cells (E), or Jurkat T cells treated as indicated (F, G) via mass spectrometry (E and G) or a spectrophotometric method (F). Jurkat cells were treated for the indicated times (F) or for 24 hr (G). NS, not significant; *p<0.05; **p<0.01; ***p<0.001 (unpaired two-tailed t-test with Welch’s (B, D, E and G) and Bonferroni correction (B and G)). Data show one experiment representative of at least three independent experiments. Error bars indicate mean ± s.e.m.

DOI: http://dx.doi.org/10.7554/eLife.14814.011

Figure 4—figure supplement 1. TCR signaling and cellular UDP-GlcNAc levels modulate the degree of poly-LacNAc induced by branching Deficiency.

(A–C) Jurkat T cells were treated with SW for the indicated times and analyzed for lectin binding by flow cytometry. (D and E) Jurkat T cells were treated as indicated for 72 hr and analyzed for LEA binding by flow cytometry. (F and G) Resting primary human T cells were treated as indicated and analyzed for LEA (F) or L-PHA (G) binding by flow cytometry. NS, not significant; *p<0.05; **p<0.01; ***p<0.001 (unpaired two-tailed t-test with Welch’s (D–G) and Bonferroni correction (D–G)). Data show one experiment representative of at least three independent experiments. Error bars indicate mean ± s.e.m.

DOI: http://dx.doi.org/10.7554/eLife.14814.012