src py416  (Cell Signaling Technology Inc)

Journal: ImmunoHorizons

Article Title: HIV Nef disrupts Lck signaling by inducing aberrant phosphorylation of its substrates

doi: 10.1093/immhor/vlaf016

Figure Lengend Snippet: Higher Lck, pLck Y394 , and DPho-Lck constitutive levels, but impaired Lck degradation and activation in stimulated Nef Tg thymocytes. Isolated total thymocytes of Nef Tg and non-Tg mice were stimulated or not with anti-CD3ε or anti-CD4 or both anti-CD3ε+anti-CD4 Abs, for different times (0 to 10 min), as indicated, at 37 °C and analyzed. (A, B) Levels of total Lck proteins (A) or active phosphorylated Lck (pLck Y394 ) (B) were determined respectively with anti-Lck (3A5) or anti-Src pY416 (upper panels) Ab by Western blotting. Membranes were stripped and reacted with anti-actin (A) or anti-Lck (B) Ab (lower panels). For quantification (graphs), the intensities of Lck and pLck Y394 were evaluated relative to those of actin or Lck, respectively, and compared to those in non-Tg mice (value = 1). Results from a single experiment are presented in panels A and B, and the same Western blot with anti-Lck Ab is shown in upper panel A and lower panel B, for clarity. Statistical comparisons were performed using the Student’s t test with data pooled from 3 independent experiments. * P < 0.05; ns, not significant. (C) Detection of Lck pY505 levels. FACS analysis of unstimulated thymocytes from non-Tg (grey) and Nef Tg (black) mice. Cells were labeled with anti-CD4 and anti-CD8 Abs for extracellular cell surface staining and with phospho-specific anti-Lck pY505 Ab for intracellular staining. Graph shows Lck pY505 -positive cells after gating on CD4 + T cells. Thymocytes from Lck-deficient non-Tg mice were used as negative controls. Note the enhanced levels of pY505 staining in Nef Tg thymocytes. Representative of 2 independent experiments. (D, E) Detection of DPho-Lck active form in Nef Tg and non-Tg thymocytes. Lck was first immunoprecipitated from unstimulated thymocyte extracts with anti-Lck, followed by a second immunoprecipitation with anti-pY505 Ab, and then analyzed in Western blot with anti-pY394 or anti-pY505 Ab and compared with 32 P-Lck levels from IVKA from the same immunoprecipitates (D). For quantification (E), the intensity of the p-Lck Y505 band was evaluated relative to that of the total Lck band (ratio p-Lck Y505 /Lck) and compared to that in non-Tg extract (value = 1). Data pooled from 3 independent experiments. Statistical comparison was performed using the Student’s t test, ** P < 0.01. (F) Lck tyrosine kinase activity in unstimulated (–) and stimulated (+) (5 min) Tg and non-Tg thymocytes. Total protein extracts were reacted with anti-Lck (3A5) Ab and immunoprecipitates used for IVKA at room temperature. Proteins were run on SDS-PAGE and transferred to membrane for detection of 32 P-labeled proteins (upper panel) and for Western blotting with rabbit polyclonal anti-Lck Ab (lower panel). Representative experiment out of 3 performed.

Article Snippet: Abs used for immunoblotting were: anti-pY (4G10) (Upstate Biotechnology), anti-Cbl (clone 7G10) (Upstate Biotechnology), anti-Lck (3A5) mAb (Santa Cruz Biotechnology, Santa Cruz, California), anti-Src pY416 (Cell Signaling Technology, Beverly, Massachusetts), anti-Src pY505 (Cell Signaling Technology, Beverly, Massachusetts), anti-Zap-70 (clone 99F2) (Cell Signaling Technology, Beverly, Massachusetts), anti-CD3ζ (6B10.2) (Santa Cruz Biotechnology, Santa Cruz, California), anti-Zap-70 pY292 (Invitrogen), anti-Zap-70 pY319 (Cell Signaling Technology, Beverly, Massachusetts), anti-Zap-70 pY493 (Cell Signaling Technology, Beverly, Massachusetts), anti-Nef clone NF2-B2 (NIH AIDS Research and Reference Reagent Program), anti-GAPDH (6C5) mAb (Abcam, Cambridge, Massachusetts), anti-actin (Sigma-Aldrich, St. Louis, Missouri), goat anti-mouse IRDye 800 (LI-COR, Lincoln, Nebraska), anti-mouse IgG (H+L) Alexa Fluor 680 and 555 (Cedarlane Laboratories) and IgG anti-rabbit (H+L) Alexa Fluor 680, 488, and 633.

Techniques: Activation Assay, Isolation, Western Blot, Labeling, Staining, Immunoprecipitation, Comparison, Activity Assay, SDS Page, Membrane

Journal: Cell reports

Article Title: Blood flow patterns switch VEGFR2 activity through differential S-nitrosylation and S-oxidation

doi: 10.1016/j.celrep.2023.113361

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: c-SRC (pY416) , Cell Signaling Technology , Cat# 2101; RRID:AB_331697.

Techniques: Virus, Recombinant, Biotin Switch Assay, Mutagenesis, Control, Sequencing, Software