Journal: Frontiers in immunology
Article Title: A non-invasive nanobody probe for high precision mapping of Lck spatial distribution.
doi: 10.3389/fimmu.2024.1440499
Figure Lengend Snippet: FIGURE 2 NbGT is a is a highly precise probe for Lck in intact cells. (A) Schematic representation of N-terminally modified NbGT constructs used in this study. Myristoylated (Glycine, G) and Palmitoylated (Cysteine, C) residues are in bold and underlined. (B, C) N-terminal modifications do not compromise NbGT binding to Lck. (B) Constitutively expressed Lck and the indicated N-terminally modified NbGT constructs were transiently cotransfected in HEK293 cells. Postnuclear lysates were subjected to immunoprecipitation as in Figure 1A. Western blots of total cell lysates ensure equal expression of NbGT and Lck constructs in all samples. (C) Representative confocal microscopy imaging of HEK293 cells co-transfected with Lck (red) and CD4- NbGT (green) as indicated. Lck staining of Jurkat cells right-hand side panel serves as a means of comparison of the subcellular distribution between endogenous (Jurkat) and exogenously overexpressed proteins (HEK 293). Scale bar, 10µm. (D) Expression of N-terminally modified NbGT constructs in Jurkat cells does not affect Lck regulation. Stable Jurkat cell lines expressing the indicated N-terminally modified NbGT forms and their -Dox counterparts were stained with antibodies against phosphorylated forms of the activating (Y394) and inhibitory (Y505) tyrosines and analyzed by FACS. MFI values for each antibody staining were determined by analysis with the Flowjo software. Graphs show collective data from three independent experiments (Unpaired Student t test; mean +/- standard deviation [SD]; ns: not significant). (E) Expression of N-terminally modified NbGT constructs in Jurkat cells does not compromise the physiological function of Lck. Stable Jurkat cell lines expressing the indicated N-terminally modified NbGT forms and their -Dox counterparts were either left untreated or were stimulated with anti-CD3ϵ for 2min at 37°C. Samples were stained with an antibody recognizing the phosphorylated form of the TCR z chain ITAM tyrosine 142 (pY142) and analysed by FACS. The Graph depicts frequency of cells positive for Y142 phosphorylation from three independent experiments (Unpaired Student t test; mean +/- SD; ns: not significant). (F) N-terminally modified NbGT constructs colocalize with endogenous Lck. Jurkat cells expressing the indicated N-terminally modified NbGT forms were immobilized on poly-D-lysine coated microscope slides, stained for Lck (red) and HA (green), and visualized by confocal microscopy. Colocalization of Lck and NbGT was quantified by the coloc2 pre-installed plugin of Image J Collective colocalization analysis results from two independent experiments are displayed on the adjacent graph. Number of cells for each sample ≥30. Scale bar, 5µm (Unpaired Student t test; mean +/- SD; ns, not significant, ****P < 0.0001). (G) Gradation of Lck binding amongst the different N-terminally modified NbGT constructs. Post nuclear lysates of Jurkat cells expressing the indicated N-terminally modified NbGT forms and their -Dox counterparts were subjected to immunoprecipitation using anti-HA beads and Nb-bound Lck was revealed by western blotting as in Figure 1A. Western blots of total cell lysates ensure equal expression of Lck and NbGT constructs in all samples. Immunoprecipitation potency was quantitated by densitometric analysis of the immunoprecipitated Lck bands, normalized for individual Nb expression levels as revealed by a-HA blots. Collective data from 4 independent experiments is depicted in the adjacent bar graph. (Unpaired Student t test; mean +/- SD; **P < 0.01, ****P < 0.0001).
Article Snippet: HA-Tag (C29F4) Rabbit mAb (Sepharose® Bead Conjugate) (Cat: 3956) used for Immunoprecipitation, Mouse anti-HA (Cat: 2367) used for western blotting, Rabbit anti-pSrc (Y416), detecting Lck pY394 (Cat: 2101), Rabbit anti-phospho-Lck (Y505) (Cat: 2751) antibodies were purchased from Cell Signaling Technology (CST).
Techniques: Construct, Binding Assay, Immunoprecipitation, Western Blot, Expressing, Confocal Microscopy, Imaging, Transfection, Staining, Comparison, Software, Standard Deviation, Phospho-proteomics, Microscopy
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![FIGURE 2 NbGT is a is a highly precise probe for Lck in intact cells. (A) Schematic representation of N-terminally modified NbGT constructs used in this study. Myristoylated (Glycine, G) and Palmitoylated (Cysteine, C) residues are in bold and underlined. (B, C) N-terminal modifications do not compromise NbGT binding to Lck. (B) Constitutively expressed Lck and the indicated N-terminally modified NbGT constructs were transiently cotransfected in HEK293 cells. Postnuclear lysates were subjected to immunoprecipitation as in Figure 1A. Western blots of total cell lysates ensure equal expression of NbGT and Lck constructs in all samples. (C) Representative confocal microscopy imaging of HEK293 cells co-transfected with Lck (red) and CD4- NbGT (green) as indicated. Lck staining of Jurkat cells right-hand side panel serves as a means of comparison of the subcellular distribution between endogenous (Jurkat) and exogenously overexpressed proteins (HEK 293). Scale bar, 10µm. (D) Expression of N-terminally modified NbGT constructs in Jurkat cells does not affect Lck regulation. Stable Jurkat cell lines expressing the indicated N-terminally modified NbGT forms and their -Dox counterparts were stained with antibodies against phosphorylated forms of the activating (Y394) and inhibitory <t>(Y505)</t> tyrosines and analyzed by FACS. MFI values for each antibody staining were determined by analysis with the Flowjo software. Graphs show collective data from three independent experiments (Unpaired Student t test; mean +/- standard deviation [SD]; ns: not significant). (E) Expression of N-terminally modified NbGT constructs in Jurkat cells does not compromise the physiological function of Lck. Stable Jurkat cell lines expressing the indicated N-terminally modified NbGT forms and their -Dox counterparts were either left untreated or were stimulated with anti-CD3ϵ for 2min at 37°C. Samples were stained with an antibody recognizing the phosphorylated form of the TCR z chain ITAM tyrosine 142 (pY142) and analysed by FACS. The Graph depicts frequency of cells positive for Y142 phosphorylation from three independent experiments (Unpaired Student t test; mean +/- SD; ns: not significant). (F) N-terminally modified NbGT constructs colocalize with endogenous Lck. Jurkat cells expressing the indicated N-terminally modified NbGT forms were immobilized on poly-D-lysine coated microscope slides, stained for Lck (red) and HA (green), and visualized by confocal microscopy. Colocalization of Lck and NbGT was quantified by the coloc2 pre-installed plugin of Image J Collective colocalization analysis results from two independent experiments are displayed on the adjacent graph. Number of cells for each sample ≥30. Scale bar, 5µm (Unpaired Student t test; mean +/- SD; ns, not significant, ****P < 0.0001). (G) Gradation of Lck binding amongst the different N-terminally modified NbGT constructs. Post nuclear lysates of Jurkat cells expressing the indicated N-terminally modified NbGT forms and their -Dox counterparts were subjected to immunoprecipitation using anti-HA beads and Nb-bound Lck was revealed by western blotting as in Figure 1A. Western blots of total cell lysates ensure equal expression of Lck and NbGT constructs in all samples. Immunoprecipitation potency was quantitated by densitometric analysis of the immunoprecipitated Lck bands, normalized for individual Nb expression levels as revealed by a-HA blots. Collective data from 4 independent experiments is depicted in the adjacent bar graph. (Unpaired Student t test; mean +/- SD; **P < 0.01, ****P < 0.0001).](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_1567/pm39021567/pm39021567__page4_image1.jpg)