anti src py416  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: Role of the membrane anchor in the regulation of Lck activity

doi: 10.1016/j.jbc.2022.102663

Figure Lengend Snippet: Dynamic maintenance of the Lck A pool . A , schematics of the generation and maintenance of Lck isoforms at the PM. From left to right : inactive (Lck I ), primed (Lck P ), active (Lck A ), active-double phosphorylated (Lck ADP ). CD45 is in large stoichiometric excess (>>) over Lck. B , Left , 3D-SIM of Lck ( green ) in CD4 + T cells or JCaM1.6 cells expressing Lck or LckΔSH4. Scale bars ( white ). PM and nucleus are neatly defined by CD45 ( red ) and DAPI staining ( blue ), respectively. Right , histograms of the ratio of Lck or LckΔSH4 amounts detected at PM and in CP (PM/CP). Error bars: SD for n ≥ 10 cells of three or more independent experiments. Unpaired t test: p > 0.5 (non-significant, ns) for CD4 + T cells versus JCaM1.6-Lck; ∗∗∗∗ p < 0.0001 for CD4 + T cells versus LckΔSH4. C , Left , 3D-SIM of pY394-Lck ( green ) in CD4 + T cells or in JCaM1.6 expressing Lck. Right , histograms of PM/CP ratio of pY394 in CD4 + T cells or in JCaM1.6 expressing Lck. Error bars: SD for n ≥ 10 cells from three or more independent experiments. Unpaired t test, ∗∗∗∗ p < 0.0001. D , Left , representative FCM of Lck A in Cln20 cells treated ( red ) with 2 μM A770041 or carrier (DMSO, blue ) at 37 °C for 30 s or 5 min. JCaM1.6 ( gray ), negative control to set pY416 antibody (Ab) background. Right, histogram of mean ± SD of Lck A (% of inhibition), n = 3. Unpaired t test, ∗∗∗∗ p < 0.0001. E , Left , representative FCM of Lck A in Clone 20 cells reacted ( green ) or not ( blue ) with 100 μM catalase-treated pervanadate (PV) at 37 °C for 1 min. JCaM1.6 ( gray ), negative control for pY416 Ab background. Right , histogram of mean ± SEM of Lck A n = 2, unpaired t test, ∗∗ p < 0.01. F , Left , representative FCM of pY505-Lck in Jurkat cells treated ( red ) with 5 μM A770041 or carrier (DMSO, blue ) at 37 °C for 5 min. JCaM1.6 ( gray ) negative control for pY505-Lck Ab background. Right , histogram of mean ± SD of Lck A (% of inhibition), n = 4, unpaired t test, ∗∗∗∗ p < 0.0001. G , Left , 3D-SIM of pY505-Lck ( green ) in CD4 + T cells or in JCaM1.6 expressing Lck or LckΔSH4. Right , histogram of PM/CP ratio for pY505 in CD4 + T cells or in JCaM1.6 expressing Lck or LckΔSH4. Error bars: SD for n ≥ 10 cells from three or more independent experiments, p > 0.5 (non-significant, ns). 3D-SIM, 3D structured illumination microscopy; CP, cytoplasmic; FCM, flow cytometry; Lck A , active form of Lck; PM, plasma membrane; LckΔSH4, Lck-lacking SH4.

Article Snippet: Rabbit anti-Lck mAb-PE (73A5) mAb, rabbit anti-pY505-Lck (#2751), and rabbit anti-pY416-Src (#2101) polyclonal Abs were from Cell Signaling Technology.

Techniques: Expressing, Staining, Negative Control, Inhibition, Microscopy, Flow Cytometry, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Role of the membrane anchor in the regulation of Lck activity

doi: 10.1016/j.jbc.2022.102663

Figure Lengend Snippet: Lck A dependence on Lck T . A , schematics of simultaneous detection of Lck T and Lck A by anti-Lck (73A5) Ab ( red ) and anti-pY416 Ab ( blue ), respectively by FCM. 73A5 Ab recognizes an epitope at Lck C-terminal sequence ( Fig. S2 A ) displayed by Lck I , Lck P , and Lck A ( Fig. S2 , B and C ). Note that 73A5 and anti-pY416 Abs do not hinder each other’s binding ( Fig. S2 D ). B , flow chart of the experimental procedure for assessing Lck A dependence on Lck T . Left , representative 2D FCM plot of Cln20 stained with Lck A and Lck T . Middle, Conversion of × (Lck T ) and y (Lck A ) axes from a logarithmic to a linear scale and a dense binning (n = 73) applied to Lck T values in the Lck T axes. Geometric median for Lck A and Lck T in each bin were calculated. Right, background-subtracted values of the geometric median for Lck A and Lck T in each bin were subjected to nonlinear regression analysis. Nonlinear regression fit of Lck A (MFI - Bkg) versus Lck T (MFI - Bkg), n = 2, R 2 = 0.99; F-test p < 0.0001. C , reactions considered for the probabilistic model of Lck A formation. The model refers to PM-resident Lck. Reaction (1) indicates the dominant effect of CD45 over Csk (as deduced by our data) to maintain low steady levels of Lck P . P PA and P AA are the probabilities of generating Lck A from the reactions: Lck P + Lck P and Lck P + Lck A , respectively. See Main Text and for further details on the basis of the empirical model. D , the increase of Lck A as a function of Lck T obtained by changing at random P PA and P AA for reactions (2) and (3) showed in ( C ). The line of best fit of the empirical model of the experimental data was obtained for the values of P PA and P AA indicated in the inset. F-test p < 0.00001. E , schematics of the “Lck cycle” at the PM, where Lck A is generated and maintained by the antagonism between CD45 and Lck for phosphorylation at Y394. Lck I is rapidly dephosphorylated at Y505 by CD45 and converted into Lck P . Lck P in turn generates Lck A by two independent reactions: Lck P + Lck P or Lck A + Lck P pair, as suggested in ( C ). The likelihood of Lck A to be dephosphorylated or not by CD45 depends on the membrane lipid environment in which Lck A dynamically resides. The gray halo represents a- L o membrane nanodomain (or raft). Abs, antibodies; FCM, flow cytometry; Csk, C-terminal Src kinase; Lck A , active form of Lck; MFI, median fluorescence intensity; PM, plasma membrane.

Article Snippet: Rabbit anti-Lck mAb-PE (73A5) mAb, rabbit anti-pY505-Lck (#2751), and rabbit anti-pY416-Src (#2101) polyclonal Abs were from Cell Signaling Technology.

Techniques: Sequencing, Binding Assay, Staining, Generated, Phospho-proteomics, Membrane, Flow Cytometry, Fluorescence, Clinical Proteomics