Journal: Science Advances

Article Title: mDia1/3-dependent actin polymerization spatiotemporally controls LAT phosphorylation by Zap70 at the immune synapse

doi: 10.1126/sciadv.aay2432

Figure Lengend Snippet: ( A ) Relative expression of the mDia isoforms in naïve CD8 T cells as determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). mDia1 and mDia3 levels are normalized to that of mDia2. Bars represent means ± SEM ( n = 3). ( B ) Scheme of the experimental schedule for the conditional KO of mDia1 and mDia3 in UBC-creERT2 × mDia1/3 double-floxed mice by tamoxifen injection. Mice received intraperitoneal (i.p.) tamoxifen injections consecutively for 5 days. Spleen and peripheral lymph nodes were isolated at 2 or 3 days after the last tamoxifen injection and naïve CD8 T cells were then purified by magnetic-activated cell sorting (MACS). ( C ) Representative Western blotting data of mDia1 and mDia3 protein levels in the lysates of control mDia1/3 double-floxed and tamoxifen-induced mDia1/3 conditional DKO (cDKO) naïve CD8 T cells. ( D ) Western blotting analysis of mDia1 and mDia3 protein levels and total and phosphorylated protein levels of pZap70 [Y319] and pLAT [Y191] in the lysates of control mDia1/3 double-floxed and tamoxifen-induced mDia1/3 cDKO naïve CD8 T cells unstimulated (0) or stimulated for 1.5, 3, or 10 min with anti-CD3 (10 μg/ml) and anti-CD28 (2 μg/ml) antibodies. Data are representative of three independent experiments. ( E ) Densitometric quantification of the Western blotting data in (D). The value of the phosphorylated form relative to total protein was normalized to that obtained for unstimulated control cells. ( F ) ELISA analysis of IL-2 levels secreted by control mDia1/3 double-floxed and mDia1/3 cDKO naïve CD8 T cells after stimulation for 48 hours in vitro without (0) or with anti-CD3 antibody (1, 2, 4, or 10 μg/ml) and anti-CD28 antibody (2 μg/ml). n = 4 for each group. Error bars represent SD. ( G ) Anti-CD3/CD28–induced proliferation of naïve CD8 T cells from control mDia1/3 double-floxed and mDia1/3 cDKO mice. Naïve CD8 T cells were seeded in 96-well plates coated with anti-CD3 antibody (0, 1, 2, 4, or 10 μg/ml) and in the presence of soluble anti-CD28 antibody (2 μg/ml). Cells were cultured for 48 hours and then assayed for proliferation. n = 4. Error bars represent SD.

Article Snippet: Naïve CD8 T cells from control or tamoxifen-induced mDia1/3 conditional DKO mice were isolated using a negative selection MACS isolation kit (130-096-543, Miltenyi Biotec).

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Injection, Isolation, Purification, FACS, Western Blot, Control, Enzyme-linked Immunosorbent Assay, In Vitro, Cell Culture