Journal: Journal of Clinical Immunology

Article Title: A Novel Biallelic LCK Variant Resulting in Profound T-Cell Immune Deficiency and Review of the Literature

doi: 10.1007/s10875-023-01602-8

Figure Lengend Snippet: Reduced protein expression and TCR signaling due to LCK C465R. A LCK and GAPDH immunoblots of whole cell lysates of patient or healthy control (HC) T-cell blasts unstimulated or stimulated with anti-CD3 (2 min), anti-CD3/CD28 (2 min), PMA/ionomycin (P/I, 5 min) or treated with the selective LCK-inhibitor A770041 (A77, 10 min). Orange arrowheads indicate LCK bands. B pZAP70, pSFK (pY416), pERK1/2 or GAPDH immunoblots, conditions as in A . Red arrowheads indicate pLCK bands, black arrowheads pFYN-T. Numbers below pERK1/2 blot indicate fold change pERK1/2 band intensity (HC unstimulated =1), intensity normalized to GAPDH loading (numbers below GAPDH blot; GAPDH signal intensity normalized to HC unstimulated) C LCK, HA-tag and GAPDH immunoblots of whole cell lysates of Jurkat E6.1, J.CaM1.6, and stable transduced cells line expressing either LCK WT or LCK C465R with or without on C-terminal HA-tag or transduced with pCDH containing only the expression cassette for the extracellular domain of the low-affinity nerve growth factor receptor (LNGFR) (J.CaM EV). D Overlay of Ca 2+ -flux measurement in the indicated cell lines. Experiment representative of 3 independent experiments. E pZAP70, pSFK (pY416), pERK1/2 immunoblots in J.CaM1.6 cells transduced with pLJM1 containing LCK WT or LCK C465R and stimulated with either anti-CD3 (2, 5 or 15 min), anti-CD3/CD28 (2 or 5 min), PMA/ionomycin (5 min) or left untreated

Article Snippet: After a blocking step with 3% BSA TBS-T, immunoblotting was performed with the following antibodies: mouse anti-human FYN (clone E-3), mouse anti-human GAPDH (clone 6C5), mouse anti-human LCK (clone 3A5), mouse-IgGκ BP-HRP, rabbit-IgGκ BP-HRP, anti-mouse-IgG-HRP (all Santa Cruz Biotechnology) or rabbit anti-human pZAP70 pY319 (clone 65E4), rabbit anti-human polyclonal pSRC-family kinase (pSFK) pY416, rabbit anti-human pERK1/2 pT202/pY204 (clone 197G2), and rabbit anti-HA-tag (clone C29F4) (all Cell Signaling Technologies).

Techniques: Expressing, Western Blot, Control, Transduction

Journal: Nature Communications

Article Title: Lyn kinase regulates egress of flaviviruses in autophagosome-derived organelles

doi: 10.1038/s41467-020-19028-w

Figure Lengend Snippet: a BHK21 and Vero E6 cells were infected with Dengue virus at varying MOI (0.1, 1.0, 5 and 10) for 24 h. Lysates prepared from infected cells were immunoblotted with antibodies against phosphorylated SFKs, total SFK and anti-flavivirus envelope 4G2 antibodies to detect virus particles. Gapdh was used as loading control. b Densitometric analyses were performed on immunoblots. Data are presented as fold change of pSFK expression normalised to total SFK compared to mock-infected cells (mean ± s.d of five independent experiments). c Cells were infected with Zika virus at varying MOI of 0.1, 1 and 5. At indicated time intervals post infection, lysates prepared from mock and infected cells were immunoblotted with anti-pSFK antibodies, anti-SFK and 4G2 antibodies to detect virions. Gapdh levels were measured as loading control. d Densitometric analyses were performed on immunoblots from four independent experiments. Data are presented as fold change of pSFK/total SFK of each sample compared to mock-infected cells set at 1. Error bars represent mean ± s.d. from four independent experiments. e Cells constitutively secreting either Zika (left panel) or Dengue (middle panel) VLPs were immunoblotted to detect phosphorylated SFKs and total SFK. Cells expressing the secretory reporter proteins ssHRP KDEL or ssHRP were measured as positive and negative controls for SFK activation (right panel). f Densitometric analyses were performed on immunoblots from five to six independent experiments (Zika and Dengue) and three independent experiments for ssHRP. Data are presented as fold change of pSFK/total SFK for each sample compared to control cells set at 1. Error bars represent mean ± s.d. from five independent experiments (Zika and Dengue) and three independent experiments (ssHRP).

Article Snippet: For biochemical analyses, the following antibodies were used: mouse anti-E mAb 4G2 (dilution 1:1000) from Novus Biologicals, or prepared using hybridoma cells D1-4G2-4-15 from ATCC; rabbit anti-phosphorylated Src family mAb (1:1200), rabbit anti-Lyn mAb (1:1500), rabbit anti-Src mAb (1: 1000) and rabbit anti-Fyn (1:1000) from Cell Signaling Technology; mouse anti-Gapdh mAb (1:2000) from Abcam.

Techniques: Infection, Virus, Control, Western Blot, Expressing, Activation Assay