src kinase inhibitor (ski-1  (Millipore)

Journal: Journal of Inflammation (London, England)

Article Title: Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

doi: 10.1186/1476-9255-3-8

Figure Lengend Snippet: Inhibition by PP2 or SKI-1 of zymosan- and S.aureus -induced release of arachidonate . Macrophages were labeled with [3H]arachidonic acid for 20 h. The cells were pretreated for 15 min with the indicated concentrations of PP2 (A) or SKI-1 (B) followed by stimulation with either zymosan (●) for 45 min or S.aureus (▲) for 60 min. Results are mean ± SEM (n = 3) and corrected for the release in control cultures.

Article Snippet: LFM-A13 and Src kinase inhibitor I (SKI-1) from Calbiochem (La Jolla, CA, USA).

Techniques: Inhibition, Labeling

Journal: Journal of Inflammation (London, England)

Article Title: Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

doi: 10.1186/1476-9255-3-8

Figure Lengend Snippet: Inhibition by PP2 or SKI-1 of zymosan- and S.aureus - induced phosphorylation of MAP kinases . Macrophages were pretreated for 15 min with PP2 (1–10 μM), followed by stimulation with zymosan ( A ) or S.aureus ( B ) for 20 min. ( C ) Macrophages were pretreated for 15 min with SKI-1 (5 μM), followed by stimulation with zymosan or S.aureus for 20 min. Equal amounts of cell lysate were run on 10% polyacrylamide gels and probed with phosphospecific antibodies against ERK, p38 and JNK. The membrane was reprobed with ERK-2 antibody to verify equal loading of protein.

Article Snippet: LFM-A13 and Src kinase inhibitor I (SKI-1) from Calbiochem (La Jolla, CA, USA).

Techniques: Inhibition

Journal: Journal of Inflammation (London, England)

Article Title: Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

doi: 10.1186/1476-9255-3-8

Figure Lengend Snippet: Zymosan but not S.aureus induced tyrosine phosphorylation of PLC γ2 . ( A ) Macrophages were stimulated with zymosan(z) or S.aureus ( S.a ) for 45 min. (B and C) Macrophages were pretreated for 15 min with either PP2 (5 μM), wortmannin (W, 100 nM) ( B ) or SKI-1 (5 μM) ( C ) followed by stimulation with zymosan for 30 min. Cell lysates were immunoprecipitated with antibody against PLCγ2 as described, followed by Western blot analysis with phosphotyrosine-specific antibody. The membrane was stripped and reprobed with antibody against PLCγ2. The data are representative of three separate experiments.

Article Snippet: LFM-A13 and Src kinase inhibitor I (SKI-1) from Calbiochem (La Jolla, CA, USA).

Techniques: Immunoprecipitation, Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

doi: 10.1186/1476-9255-3-8

Figure Lengend Snippet: Zymosan and bacteria but not LPS or peptidoglycan (PGN) induce phosphorylation of Btk . ( A ) Macrophages were stimulated with zymosan (zym), S.aureus ( S.a .), LPS or PGN for 30 min. (B and C) Macrophages were pretreated for 15 min with either wortmannin (W, 100 nM), PP2 (5 μM), SU6656 (5 μM) or SKI-1 (5 μM), followed by stimulation with zymosan ( B ) or S.aureus ( C ) for 30 min. Equal amounts of cell lysate were run on polyacrylamide gels and probed with phosphospecific antibodies against Btk. The membrane was reprobed with ERK-2 antibody to verify equal loading of proteins. The data are representative of three separate experiments.

Article Snippet: LFM-A13 and Src kinase inhibitor I (SKI-1) from Calbiochem (La Jolla, CA, USA).

Techniques:

Journal: Oncogenesis

Article Title: Squalene synthase promotes the invasion of lung cancer cells via the osteopontin/ERK pathway

doi: 10.1038/s41389-020-00262-2

Figure Lengend Snippet: a CL1-0 cells infected with the vector and SQS overexpression plasmid were subjected to western blot analyses of SQS, pSrc, Src, pERK, ERK, pAKT 473 , AKT and α-tubulin levels. b Comparison of pSrc, Src, pERK, ERK, pAKT 473 , and AKT levels between SQS knockdown A549 and CL1-5 cells and nonsilenced shRNA control cells. c Western blots showing the levels of pSrc, Src, pERK, ERK, pAKT 473 , AKT and α-tubulin in CL1-0/SQS cells after treatment with Src kinase inhibitor-1 (SKI, 10 μM). d Heatmap showing the protein levels of AKT, AKT_pS473, Src, and Src_pY416 and migration ability in the lung cancer cell panel. e Plot showing the correlations between the protein levels of AKT_pS473 protein/Src_pY416 protein and migration in the lung cancer cell panel. f CL1-0/SQS cells were treated with or without DMSO, Src inhibitor-1 (SKI) (10 μM), PD98059 (20 μM) and LY294002 (10 μM). Top panel, The total protein samples were subjected to western blot analyses of SQS and α-tubulin levels (cell extract, CE). Middle panel, MMP1 protein expression levels from condition medium (CM). Bottom panel, Invasion (open) and migration (filled) of CL1-0/SQS cells treated with DMSO, SKI, PD98059 and LY294002 compared to CL1-0/Vector cells. Data are presented as the means ± SDs. ** P < 0.01. g CL1-0/SQS cells were treated with or without MβCD (10 mM) for 30 min 37 °C. Western blot analyses of SQS, pSrc, pERK and pAKT and α-tubulin levels of CL1-0/SQS cells. h Effect of cholesterol replenishment on the phosphorylation of Src, ERK, and AKT in CL1-0 cells.

Article Snippet: Src Inhibitor-1 (SKI) (Cat. S2075, 10 μM), PD-98059 (a mitogen-activated protein (MAP) kinase inhibitor, Cat. P215, 20 μM) and cholesterol (Cat.C3045) were purchased from Sigma, St. Louis, MO, USA.

Techniques: Infection, Plasmid Preparation, Over Expression, Western Blot, shRNA, Migration, Expressing

Journal: PLoS ONE

Article Title: The Ubiquitin Ligase CBLC Maintains the Network Organization of the Golgi Apparatus

doi: 10.1371/journal.pone.0138789

Figure Lengend Snippet: Giantin staining in HeLa cells. Fragmentation Index measured in quadruplicates on at least 400 cells per condition. Error bars show SD statistical significance (p) measured by unpaired Student’s t-test. (*) represents p<0.05, (**) represents p<0.01 and (***) represents p<0.001. (A and D) Co-transfection of CBLC and SRC siRNAs in HeLa cells (B and E) Treatment of CBLC-depleted cells with SRC inhibitors PP2 or SKI-1 rescues Golgi morphology. (C and F) Co-transfection of CBLC and ARF1 siRNAs in HeLa cells rescues Golgi morphology. Scale bar = 10 μm.

Article Snippet: After 48 hours of siRNA transfection, medium was aspirated, replaced with fresh medium containing 5 μM SRC inhibitors PP2 or SRC inhibitor kinase 1 SKI-1 (Sigma) and incubated at 37°C in a 10% CO 2 humidified incubator for a further 24 hours before fixation.

Techniques: Staining, Cotransfection

Journal: Cardiovascular Research

Article Title: Extracellular component hyaluronic acid and its receptor Hmmr are required for epicardial EMT during heart regeneration

doi: 10.1093/cvr/cvv190

Figure Lengend Snippet: FAK and Src inhibition suppressed angiogenesis in regenerating hearts. (A–C) At 30 dpa, hearts injected for 10 days with PF-573228, a FAK inhibitor (B) (n = 10), or with SKI-1 (C) (n = 13), a Src inhibitor, failed to properly regenerate the heart compared with DMSO controls (A) (n = 31). Red arrows delineate scar. (D) Graph showing clot area at 30 dpa after injections of PF-573228 or SKI-1. (E–M) PF-573228 and SKI-1 injections inhibited angiogenesis. (E–G) AFOG staining of hearts at 10 dpa, injected with DMSO (E) (n = 15), PF-573228 (F) (n = 10), or SKI-1 (G) (n = 10). (H–M) Tg(fli1a:EGFP) hearts injected with DMSO (H and K), PF-573228 (I and L), or with SKI-1 (J and M), immunostained for MHC to detect resection plane. White arrows indicate new vessels formed inside the clot. (N) Quantification fli1a+:EGFP area in the clot at 10 dpa. FAK and Src inhibition significantly reduced new vessel formation. Black boxes delimitate the areas of confocal imaging. Black bars in graphs indicate the mean and error bars the SEM. Scale bars, 100 μm. *P < 0.05; **P < 0.01; ***P < 0.001 one-way ANOVA.

Article Snippet: To suppress Src Kinase, 3 µL of 200 µM Src Inhibitor 1 (SKI-1) (Sigma) or vehicle DMSO was injected.

Techniques: Inhibition, Injection, Staining, Imaging

Journal: Cardiovascular Research

Article Title: Extracellular component hyaluronic acid and its receptor Hmmr are required for epicardial EMT during heart regeneration

doi: 10.1093/cvr/cvv190

Figure Lengend Snippet: HA and its receptor Hmmr are necessary for epicardial cell migration in ex vivo culture. (A–D) Ex vivo epicardial migration assay showing decreased Wt1b:EGFP+ migratory behaviour after hmmrVMO (B) (n = 8) and PF-573228 (D) (n = 7) treatments compared with controls (A) (n = 14) and (C) (n = 7). Images were captured at 3 days post-extraction of the heart. The migration of Wt1b:EGFP+ epicardial cells (red arrows) was measured from Day 1 to Day 3. In control PBS, DMSO, or hmmr-MutMO treatments, epicardial cells migrated on the fibrin-coated plates. In contrast, treatment with hmmrVMO, PF-573228 or SKI-1 significantly suppressed epicardial cell migration. (E–H) Graphs showing migration of Wt1b:EGFP+ cells under various treatments, including suppressing HA production in the hearts with HMC (E) (n = 13), hmmrVMO (F) (n = 8), PF-573228 (G) (n = 7), and with SKI-1, the Src kinase inhibitor (H) (n = 8). Black bars in graphs indicate the mean and error bars the SEM. Scale bars, 250 μm. *P < 0.05; **P < 0.01; ***P < 0.001. Two-way ANOVA.

Article Snippet: To suppress Src Kinase, 3 µL of 200 µM Src Inhibitor 1 (SKI-1) (Sigma) or vehicle DMSO was injected.

Techniques: Migration, Ex Vivo