rabbit anti p btk tyr223 monoclonal antibody  (Cell Signaling Technology Inc)

Journal: Blood Science

Article Title: Zanubrutinib enhances CD19 CAR T killing of B-cell lymphoma by inhibiting BTK phosphorylation, regulating PI3K/AKT/mTOR pathway, and promoting autophagy

doi: 10.1097/BS9.0000000000000276

Figure Lengend Snippet: Effect of zanubrutinib on the BTK/P110 pathway in B-cell lymphoma cells in a CAR T-cell co-culture system. (A) Protein expression statistics of BTK, p-BTK, P110-α, P110-β, P110-γ, and P110-δ in cells. (B) Protein expression of BTK, p-BTK, P110-α, P110-β, P110-γ, and P110-δ determined by western blot (n = 3). Compared with the 1/2Daudi + CD19 CAR T group or Daudi + CD19 CAR T group, * p < 0.050, ** p < 0.010. BTK = Bruton tyrosine kinase, CAR = chimeric antigen receptor.

Article Snippet: The cells were closed with bovine serum (BOSTER, Wuhan, China) for 20 minutes at room temperature, and then the ki-67 primary antibody (Proteintech, Wuhan, China) or a mixture of the LC3II primary antibody (Abcam, UK) and the p-BTK primary antibody (Proteintech) was added and incubated at 4°C overnight.

Techniques: Co-Culture Assay, Expressing, Western Blot

Journal: Blood Science

Article Title: Zanubrutinib enhances CD19 CAR T killing of B-cell lymphoma by inhibiting BTK phosphorylation, regulating PI3K/AKT/mTOR pathway, and promoting autophagy

doi: 10.1097/BS9.0000000000000276

Figure Lengend Snippet: Effect of zanubrutinib on the protein kinase B (AKT)/mechanistic target of rapamycin (mTOR)-mediated autophagy pathway in B-cell lymphoma cells in a CAR T-cell co-culture system. (A) Protein expression of AKT, p-AKT, mTOR, p-mTOR, LC3II/LC3I, and Beclin1 was measured by western blot (n = 3). (B) Co-expression of p-BTK with LC3II detected by immunofluorescence staining (20×, scale bar, 50 μm). (C) Fluorescence intensity of p-BTK with LC3II (n = 3). (D) Transmission electron microscopy (20,000×, scale bar, 500 nm, red arrows: mitochondria or mitochondrial swelling, blue arrows: rough endoplasmic reticulum, green arrows: autophagosomes and autophagolysosomes, n = 3). Compared with the 1/2Daudi + CD19 CAR T group or Daudi + CD19 CAR T group, * p < 0.050, ** p < 0.010. BTK = Bruton tyrosine kinase, CAR = chimeric antigen receptor.

Article Snippet: The cells were closed with bovine serum (BOSTER, Wuhan, China) for 20 minutes at room temperature, and then the ki-67 primary antibody (Proteintech, Wuhan, China) or a mixture of the LC3II primary antibody (Abcam, UK) and the p-BTK primary antibody (Proteintech) was added and incubated at 4°C overnight.

Techniques: Co-Culture Assay, Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence, Transmission Assay, Electron Microscopy

Journal: Blood Science

Article Title: Zanubrutinib enhances CD19 CAR T killing of B-cell lymphoma by inhibiting BTK phosphorylation, regulating PI3K/AKT/mTOR pathway, and promoting autophagy

doi: 10.1097/BS9.0000000000000276

Figure Lengend Snippet: Zanubrutinib enhances sensitivity to CAR T-cell therapy in mice with B-cell lymphoma cells. (A) Tumor size (n = 4). (B) Tumor volume (n = 4). (C) Immunofluorescence staining of ki-67 (20×, scale bar, 50 μm) and TUNEL staining (400×, scale bar, 50 μm). (D) Fluorescence intensity of ki-67 and apoptosis in TUNEL cells (n = 3). (E) Transmission electron microscopy (20,000×, scale bar, 500 nm, red arrows: mitochondria or mitochondrial swelling, yellow arrows: rough endoplasmic reticulum, blue arrows: autophagosomes and autophagolysosomes, n = 3). (F) Protein expression statistics of Bax, Bcl-2, Caspase3, cleaved Caspase3, BTK, p-BTK, LC3II/LC3I, Beclin1, P110-α, P110-β, P110-γ, P110-δ, AKT, p-AKT, mTOR, and p-mTOR in tumor tissues. (G) Protein expression of Bax, Bcl-2, Caspase3, cleaved Caspase3, BTK, p-BTK, LC3II/LC3I, Beclin1, P110-α, P110-β, P110-γ, P110-δ, AKT, p-AKT, mTOR, and p-mTOR determined by western blot (n = 3). Compared with the model group, * p < 0.050, ** p < 0.010. Compared with the CD19 CAR T group, ## p < 0.01, # p < 0.05. BTK = Bruton tyrosine kinase, CAR = chimeric antigen receptor.

Article Snippet: The cells were closed with bovine serum (BOSTER, Wuhan, China) for 20 minutes at room temperature, and then the ki-67 primary antibody (Proteintech, Wuhan, China) or a mixture of the LC3II primary antibody (Abcam, UK) and the p-BTK primary antibody (Proteintech) was added and incubated at 4°C overnight.

Techniques: Immunofluorescence, Staining, TUNEL Assay, Fluorescence, Transmission Assay, Electron Microscopy, Expressing, Western Blot

Journal: Journal of Medicinal Chemistry

Article Title: Tunable Aromatic Sulfoxides and Sulfones as Cysteine-Targeting Warheads: Exploring the Structure–Reactivity Relationship

doi: 10.1021/acs.jmedchem.5c03536

Figure Lengend Snippet: Biological evaluation of alkyne probes in living cells. (a) Dose-dependent labeling of cellular BTK. Ramos cells were treated with 10–500 nM probe for 1 h, and the resulting cell lysates were modified by click reaction with TAMRA-N 3 . Proteins were separated by SDS-PAGE, and the gel was imaged with ChemiDoc imaging system at two channels (green LED, 605/50 filter for TAMRA, red LED 695/50 filter for MW markers). Images were merged to generate the composite image. The blue arrow indicates the BTK band. The gels were subsequently stained with SimplyBlue for total protein visualization. Full gel images are presented in Figures S13–S15 . (b) Cellular protein labeling profiles of the probes, together with ibrutinib competition, confirms BTK binding. Ramos cells were pretreated with either DMSO or 1 μM ibrutinib for 30 min, followed by treatment with 100 nM probe for 1 h. The resulting cell lysates were modified and analyzed as in (a). The blue arrow indicates the BTK band. (c) Volcano plot obtained from TMT-based quantitative proteomics analysis of the pull-down performed with Ibr-2 . Ramos cells were treated with 250 nM Ibr-2 , lysed, and conjugated to biotin-N 3 . Proteins with a log 2 fold-change >1 compared to the DMSO control and an adjusted p -value <0.05 were considered significantly enriched (highlighted in blue and annotated). An enlarged version of the volcano plot is presented in Figure S19 . (d) Ibr-2 does not affect BTK activity in Ramos cells, as measured by BTK autophosphorylation. Cells were pretreated with either DMSO or 1 μM ibrutinib for 30 min, followed by treatment with 100 nM Ibr-2 for 1 h. Thereafter, the cells were washed before BCR-stimulation with antihuman IgM (10 μg/mL) for 10 min. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antibodies against phospho-BTK (Tyr223), total BTK, and β-actin.

Article Snippet: Phosphorylated BTK was probed using a primary rabbit anti-p-BTK (Tyr223) monoclonal antibody (Cell Signaling #87141), diluted 1:1000 in 5% milk in TBS-T, at 4 °C overnight.

Techniques: Labeling, Modification, SDS Page, Imaging, Staining, Binding Assay, Quantitative Proteomics, Control, Activity Assay

Journal: Advanced Science

Article Title: Platelet Rubicon Bidirectional Regulation of GPVI and Integrin αIIbβ3 Signaling Mitigates Stroke Infarction Without Compromising Hemostasis

doi: 10.1002/advs.202507509

Figure Lengend Snippet: Rubicon‐deficient platelets exhibit enhanced GPVI‐mediated aggregation and secretion but impaired αIIbβ3 outside‐in signaling. (A) Aggregation and ATP secretion of washed platelets stimulated with indicated concentrations of collagen. Aggregation and ATP secretion (with luciferase) were assessed with a Chrono‐log lumi‐aggregometer under stirring at 1200 rpm. (B) Quantification of aggregation (left) and ATP release (right) depicted in panel A. The results are shown as median with interquartile range (two‐tailed Mann–Whitney U test). (C) Aggregation after apyrase (0.1U/mL) pre‐treatment, followed by collagen stimulation. (D) Quantification of aggregation depicted in panel C. The results are shown as median with interquartile range (two‐tailed Mann–Whitney U test). (E) Aggregation after aspirin pre‐treatment, followed by collagen stimulation. (F) Quantification of aggregation depicted in panel E. The results are shown as median with interquartile range (two‐tailed Mann–Whitney U test). (G) Western blot analysis of GPVI pathway signaling proteins after collagen stimulation. Quantification of phosphorylated PLCγ2 Y1217 , Btk Y223 , Fyn Y530 , Lyn Y507 in WT and Rubicon −/− platelets stimulated with collagen. The results are shown as median with interquartile range (Wilcoxon signed‐rank test). (H) Time‐course analysis of PLCγ2 Tyr1217 phosphorylation in WT and Rubicon −/− platelets stimulated with collagen. The results are shown as mean ± SEM (two‐tailed Mann–Whitney U test). (I) Spreading of WT and Rubicon −/− platelets on immobilized fibrinogen in the presence or absence of apyrase (0.1U/mL) or ADP (1 µ m ), scale bar: 5 µm. Quantification of the areas of spread WT and Rubicon −/− platelets. The results are shown as median with interquartile range (two‐tailed Mann–Whitney U test). (J) Platelets from WT ( n = 6) and Rubcn f/f PF4‐Cre + ( n = 7) mice were resuspended with human platelet‐poor plasma at a concentration of 4 × 10 8 mL −1 , and recombined plasma was stimulated to coagulate with thrombin (0.4 U/mL), then photographed at different time points. Statistical significance was evaluated with a two‐way ANOVA test, and the results are shown as mean ± SEM (* p < 0.05, ** p < 0.01). (K) Western blot analysis of outside‐in signaling (phosphorylated β3, AKT, PLCγ2, ERK) during platelet spreading on fibrinogen.

Article Snippet: The following antibodies were used: anti‐VPS15 (Novus Biologicals, NBP1‐30463); anti‐PIK3C3 (Cell Signaling, 4263S); anti‐BECN1 (Cell Signaling, 3738S); anti‐LC3 (clone 2G6) (nano Tools, 0260); anti‐Tubulin α (sigma, T5168); anti‐p‐Akt S473 (Cell Signaling,4060S); anti‐p‐Akt T308 (Cell Signaling, 13038S); anti‐p‐ERK (Cell Signaling, 9101S); anti‐p‐p40phox (Santa Cruz, sc33403); anti‐p‐integrin β3 (Santa Cruz, sc365679), anti‐p‐integrin β3 T747 (Santa Cruz, sc101707), anti‐p‐integrin β3 T759 (Santa Cruz, sc20235); FITC‐conjugated anti‐mouse CD41 monoclonal antibody (mAb) (BD Biosciences, MWReg30, 558040); FITC‐conjugated anti‐mouse CD42b mAb (Emfret Analytics GmbH & Co. KG, Xia.B2, M043‐0); FITC‐conjugated anti‐mouse GPVI mAb (Emfret Analytics, JAQ1, M011‐1); anti‐Rubicon antibodies (Cell Signaling, D9F7, 3412; GeneTax, GTX129096); anti‐Btk antibody(Cell Signaling, D3H5, 8547); anti‐p‐Btk Y223 antibody (Cell Signaling, D1D2Z, 87457); anti‐PLCγ2 antibody (Cell Signaling, 3872); anti‐p‐PLCγ2 Y1217 antibody (Cell Signaling, 3871T); anti‐p‐Lyn Y507 antibody (Cell Signaling, 2731T); anti‐fyn antibody (Abcam, 2A10, ab119855); anti‐p‐Fyn Y530 antibody (Abcam, ab182661); anti‐p62 antibody (Cell Signaling, D6M5X, 23214); anti‐LC3 antibody (Sigma Aldrich, L7543 and L8919); anti‐LAMP2 antibody (Proteintech, 66301).

Techniques: Luciferase, Two Tailed Test, MANN-WHITNEY, Western Blot, Phospho-proteomics, Clinical Proteomics, Concentration Assay