src family kinase inhibitor 1 tert butyl  (Selleck Chemicals)

Journal: The journal of pain

Article Title: Microglial P2Y12 Signaling Contributes to Cisplatin-induced Pain Hypersensitivity via IL-18-mediated Central Sensitization in the Spinal Cord.

doi: 10.1016/j.jpain.2023.01.005

Figure Lengend Snippet: Figure 5. Spinal blockade of the P2Y12/Src family kinase (SFK)/p38 pathway suppresses the cisplatin-induced IL-18 production. (A) IL-18 immunoreactivity was colocalized with IBA-1, (B) but not with NeuN or GFAP in the spinal dorsal horn. (C) IL-18 immunoreac- tivity was colocalized with P2Y12, p-SFK, and p-p38 in the spinal dorsal horn. Tissues were harvested on day 15 post administration. Scale bars: 50 mm and 20 mm (zoom). (D−F) Real-time qPCR analyses showed that MRS2395 (D), PP2 (E) or SB239063 (F) significantly attenuated the cisplatin-induced upregulation of IL-18 mRNA expression in the spinal dorsal horn. (G−I) Western blotting analyses showed that MRS2395 (G), PP2 (H) or SB239063 (I) significantly attenuated the cisplatin-induced upregulation of IL-18 protein expression in the spinal dorsal horn. MRS2395 (2 mg, intrathecally), PP2 (10 mg, intrathecally), PP3 (10 mg, intrathecally) or SB239063 (10 mg, intrathecally) was injected daily once, on days 12, 13, 14, and 15 after the first cisplatin injection. Tissues were harvested 4 h following the last injection on day 15. Data are shown as the mean § SEM. **P < .01, in comparison with the saline + DMSO (or PP3) group; ##P < .01, compared with the cisplatin + DMSO (or PP3) group, n = 4 per group, one-way ANOVA with the post hoc Dunnett test. Abbreviations: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; GFAP, glial fibrillary acidic protein; IBA-1, ionized cal- cium-binding adapter molecule 1; IL-18, interleukin-18; NeuN, neuronal nuclei; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean; SFK, Src family kinase.TagedEnd

Article Snippet: Src family kinase inhibitor PP2 (S7008) and P38 inhibitor SB239063 (S7741) were obtained from Selleck Chemicals (China).

Techniques: Expressing, Western Blot, Injection, Comparison, Saline, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine

Article Title: CD36 deficiency ameliorates drug-induced acute liver injury in mice

doi: 10.1186/s10020-021-00325-z

Figure Lengend Snippet: Treatment with PP2 attenuated APAP induced liver injury. A Male C57 mice were starved for 16 h and i.p. injected with/without PP2 (1.5 mg/kg) prior to APAP administration (300 mg/kg). Serum was collected after 8 h and 24 h for ALT and AST measurement. B H&E staining in liver section from the mice at 24 h after APAP injection. *P < 0.05, **P < 0.01, ***P < 0.001. N = 6 mice per group

Article Snippet: The Src family kinase selective inhibitor PP2 (Selleck, China) was injected to the mice intraperitoneally (1.5 mg/kg, dissolved in ethanol) at half an hour prior to APAP-treatment.

Techniques: Injection, Staining

Journal: Cells

Article Title: Helicobacter Pylori Targets the EPHA2 Receptor Tyrosine Kinase in Gastric Cells Modulating Key Cellular Functions

doi: 10.3390/cells9020513

Figure Lengend Snippet: EPHA2 receptor downregulation induced by H. pylori infection is preceded by receptor phosphorylation early on and is followed by lysosomal degradation in the MKN74 gastric cell line. ( a ) Tyrosine and serine897 phosphorylation of EPHA2 upon H. pylori exposure, as determined by ELISA and Western blot, respectively; *** p < 0.001, ** p < 0.01, * p ≤ 0.05, ns—not significant ( p > 0.05). ( b ) Effect of PP2 (SRC family kinase inhibitor), U0126 (MEK inhibitor), and CAY10626 (PI3Kα/mTOR inhibitor) on downregulation of EPHA2 mediated by H. pylori at 24 h by Western blot and corresponding quantifications by densitometry; **** p < 0.0001, *** p < 0.001, ** p < 0.01, ns—not significant ( p > 0.05). ( c ) Effect of SRC family kinase inhibitors (PP2 and Dasatinib inhibitors) on EPHA2-tyrosine phosphorylation 1 h after H. pylori infection, as evaluated by ELISA; **** p < 0.0001, ** p < 0.01, * p ≤ 0.05. ( d ) Effect of lysosomal (Bafilomycin A1 and Concanamycin A) and proteasomal (bortezomib) inhibitors on EPHA2 receptor downregulation induced by H. pylori at 24 h post-infection as shown by Western blot and the respective relative density expressed as the ratio of infected/uninfected cells; *** p < 0.001; ** p < 0.01; ns—not significant ( p > 0.05). One-way ANOVA with post-hoc Dunnett’s or Tukey’s test.

Article Snippet: The pharmacological inhibitors used were U0126 (Cayman chemical, MI, USA), which is a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, CAY10626 (Cayman), a dual phosphatidylinositol-3-kinase (PI3Kα) and mTOR inhibitor, and the SRC kinase family inhibitors PP2 (Cayman) and Dasatinib (Selleck Chemicals, Houston, TX, USA).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Detection of experimental and clinical immune complexes by measuring SHIP-1 recruitment to the inhibitory FcγRIIB

doi: 10.4049/jimmunol.1700832

Figure Lengend Snippet: (A-C) HEK293F cells, transiently co-transfected with CD32B-SmBiT, SHIP-1-LgBiT (N-terminus) alone (A), with CD32A (B) or ITIM-mutated CD32B-SmBiT, SHIP-1-LgBiT and CD32A (C) on d0, were stimulated with medium, IC or monomeric IgG (10µg/ml) on d1. Luminescence was measured at time 0 following substrate addition, and at 5-minute intervals. (D) HEK293F cells, transfected as in (B) were pre-treated with the Src family kinase inhibitor PP2 at the indicated concentrations, or DMSO control, on d1 following transfection, followed by stimulation with medium, IC or monomeric IgG (10µg/ml). Luminescence was measured as above. (A-D) Left - schematic diagrams; right - fold inductions, with means ± S.D. of technical replicates, representative of 3 (A), at least 3 (B) and 3 (C) independent experiments. (D) No responses to medium control or monomeric IgG were detected (data not shown). The absence of response to IC with prior 10µM PP2 treatment is representative of 3 independent experiments.

Article Snippet: The Src family kinase inhibitors PP2 and Dasatinib Monohydrate were from Selleckchem.

Techniques: Transfection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PALLD Regulates Phagocytosis by Enabling Timely Actin Polymerization and Depolymerization.

doi: 10.4049/jimmunol.1602018

Figure Lengend Snippet: FIGURE 4. PALLD promotes particle binding by regulat- ing FcgRIIA clustering. (A) NB4 NC and KD cells pretreated with ATRA for 72 h were incubated with anti-FcgRIIA Ab at 4˚C for 30 min, followed by incubation with Alexa Fluor 594 secondary Ab (red) for 20 min. The cells were then fixed and labeled with DAPI (blue). FcgRIIA clustering was observed under a microscope. The images are representative of three experiments. Original magnification 363. (B) The expression levels of FcgRIIA, FcgRIIIA, and CD11b/CD18 in NC and KD cells, as shown in (A), were evaluated by flow cytometry. (C) NB4 cells were pretreated with ATRA for 72 h, and then treated with DMSO, 5 mM PP2, or 10 nM cytochalasin D for another 15 min; FcgRIIA clustering was then observed under a microscope. Images from two representative experiments are provided. (D) NC and KD cells were pretreated for 72 h with ATRA and then treated with DMSO, 5 mM PP2, or 10 nM cytochalasin D for another 15 min, and the cell’s particulate-binding ability was then examined by flow cytom- etry. Quantification of three independent experiments was shown. (E) NB4 cells pretreated with ATRA were incubated with serum-opsonized zymosan for 15 min, fixed, labeled with c-Src (red) and PALLD (green), and observed under micros- copy. (F) Lysates of NB4 cells pretreated with ATRA for 72 h were immunoprecipitated with normal rabbit IgG or PALLD Ab. The lysates were then immunoblotted with c-Src Ab. (G) NC and KD cells pretreated with ATRA were incubated with serum-opsonized E. coli for the indicated durations. The lysates were analyzed by Western blot for phosphorylated c-Src Tyr416

Article Snippet: Src family kinase inhibitor PP2 was purchased from Selleckchem; cytochalasin D and ATRA were purchased from Sigma-Aldrich.

Techniques: Binding Assay, Incubation, Labeling, Microscopy, Expressing, Cytometry, Immunoprecipitation, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Endothelial cell‐derived matrix promotes the metabolic functional maturation of hepatocyte via integrin‐Src signalling

doi: 10.1111/jcmm.13195

Figure Lengend Snippet: EC matrix promotes hepatic metabolic functional maturation dependent on Src phosphorylation. The activities of CYP 2C9 ( A ) and CYP 3A4 ( B ) after induced by rifampicin were assessed in hASC ‐ HLC s cultured on the EC matrix, which were treated with FAK , SFK and ILK inhibitors for 15 hrs. ( C ) The protein levels of p‐Src (Tyr416) and total Src were measured by Western blot in hASC ‐ HLC s cultured on the EC matrix and the collagen I‐coated substrate for 72 hrs, or cultured on the FN ‐depleted and control EC matrix, or in α5 integrin si RNA ‐treated and control si RNA ‐treated hASC ‐ HLC s, which were cultured on the EC matrix. ( D ) The protein levels of FOXA 2, HNF 4α and PXR in hASC ‐ HLC s were examined by Western blot. Statistical significance compared to the control, * P < 0.05; ** P < 0.01. Collagen I, collagen I‐coated substrate; control, the control EC matrix; FAK , focal adhesion kinase; FN depleted, the FN ‐depleted EC matrix; ILK , integrin‐linked kinase; ITG , integrin; RLU , relative luminescent unit; SFK , Src family kinase.

Article Snippet: After a 6‐hr incubation, focal adhesion kinase (FAK) inhibitor (1 μM PF228; Selleckchem, Houston, TX, USA), Src family kinase (SFK) inhibitor (5 μM, PP2, Selleckchem) and integrin‐linked kinase (ILK) inhibitor (1 μM Cpd22, Merck Millipore, Darmstadt, Germany) were added separately for 15 hrs; then, the cells were cultured for 72 hrs, and the metabolic properties of the hASC‐HLCs were analysed further.

Techniques: Functional Assay, Phospho-proteomics, Cell Culture, Western Blot, Control