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. 2023 Sep 5;6:911. doi: 10.1038/s42003-023-05269-0

Fig. 6. INPP5E sustains efficient early proximal TCR signaling and the effector function.

Fig. 6

a Jurkat cells were transfected with either siCtrl or siINPP5E. Immunoblots were performed to analyze the phosphorylation levels of indicated proteins in Ctrl or INPP5E-knockdown Jurkat T cells activated by crosslinking with anti-CD3/CD28 for the indicated times. α-tubulin and non-phosphorylated molecules were blotted as loading controls. Representative images are shown. b Jurkat cells were transfected with either siCtrl or siINPP5E. Immunostaining of pCD3ζ-Y83 (upper) and pZAP-70-Y493 (lower) in conjugates of the control or INPP5E-knockdown Jurkat T cells and CMAC-labeled SEE-pulsed APCs. Phalloidin staining was performed to visualize F-actin. Representative images are shown. c The enrichment index of phosphorylated proteins was quantified in conjugates. N = 3. n = 183 conjugates for the siCtrl, and n = 199 conjugates for siINPP5E in pCD3ζ experiments. n = 101 conjugates for Ctrl, and n = 103 conjugates for siINPP5E in pZAP-70 experiments. Error bars indicate mean ± SD. Unpaired Student t-test. ****P < 0.0001. d Jurkat cells were transfected with either control or INPP5E-specific siRNA followed by anti-CD3/CD28 incubation. Cells were subjected to nuclear and cytosolic fractionation followed by WB analysis with antibodies as indicated. Lamin B and GAPDH was used as nuclear and cytosolic marker, respectively. e Jurkat cells were transfected with either control or INPP5E-specific siRNA followed by anti-CD3/CD28 incubation. The percentage of CD40L-expressing cells was quantified. Error bars indicate mean ± SD. Paired student t-test. P < 0.05, **P ≤ 0.005. f, g Jurkat cells were transfected with either control or INPP5E-specific siRNA. An ELISA assay showing the amount of IL-2 in the cultured supernatants in siCtrl or siINPP5E Jurkat T cells. In f, T cells were activated by SEE-pulsed APCs for 24 h. In g, T cells were activated by anti-CD3/CD28 for 24 h. The results were an average of at least three independent assays. Each assay has three technical replicates. *P < 0.05. **P ≤ 0.005. h Mouse primary pan T cells were isolated from C57bl/6 mice and transduced with shCtrl or shInpp5e retrovirus. GFP positive cells were sorted and used to measure the Il-2 mRNA level on day 4. The relative mRNA levels of Inpp5e and Il-2 were quantified from three independent experiments. Error bars indicate mean ± SD. Paired student t-test. *P < 0.05, ***P < 0.001.