sfk inhibitors ski 1  (Santa Cruz Biotechnology)

Journal: Stem cell research

Article Title: Src-family Tyrosine Kinase Activities are Essential for Differentiation of Human Embryonic Stem Cells

doi: 10.1016/j.scr.2014.09.007

Figure Lengend Snippet: A) The hES cell lines H1, H7 and H9 were maintained under feeder-free conditions on Matrigel-coated plates using mTeSR1 medium. All three lines grew as large domed colonies with well-circumscribed boundaries. B) H1 hES cells were harvested 5 days after passage for RNA isolation. SFK (Blk, Fgr, Fyn, Hck, Lck, Lyn, c-Src, c-Yes), Src-related kinase (Brk, Frk, Srm) and pluripotency marker (Oct4) expression were determined by RT-PCR with GAPDH as a positive control. Images of agarose gels of the PCR products are shown after 30 and 35 PCR cycles. C) Relative SFK (Fyn, Hck, Lck, Lyn, c-Src, c-Yes) and pluripotency marker (Oct4, Nanog) expression levels across three hES cell lines. Transcript levels for the indicated genes were determined by quantitative real-time RT-PCR for the H1, H7 and H9 hES cell lines. The results are expressed as the average fold-change in mRNA level in H7 and H9 cells relative to H1 cells ± SEM (n = 3, *p < 0.05; pairwise xed reallocation randomization test). SFK expression varied by less than 2-fold across these hES cell lines (gray area), with the exception of Hck expression in H7 cells.

Article Snippet: The SFK inhibitors SKI-1 and PP2 were purchased from EMD-Millipore-Calbiochem while A-419259 was purchased from Santa Cruz Biotechnology.

Techniques: Isolation, Marker, Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Quantitative RT-PCR

Journal: Stem cell research

Article Title: Src-family Tyrosine Kinase Activities are Essential for Differentiation of Human Embryonic Stem Cells

doi: 10.1016/j.scr.2014.09.007

Figure Lengend Snippet: Lysates were prepared from mouse ES cells (D3 line), human ES cells (H1 and H7 lines) and 6-day EBs derived from them. Endogenous SFKs (c-Src, c-Yes, Fyn, Lck, Hck, Lyn) were immunoprecipitated and blotted with a phosphospecific antibody for the active form of each kinase (pY418 antibody, which recognizes the conserved phosphotyrosine residue in the activation loop of the kinase domain) as well as the respective SFK protein. Cell lysates were also blotted for Oct4 as marker of ES cell self-renewal plus actin as a loading control. This experiment was repeated twice with comparable results; a representative example is shown.

Article Snippet: The SFK inhibitors SKI-1 and PP2 were purchased from EMD-Millipore-Calbiochem while A-419259 was purchased from Santa Cruz Biotechnology.

Techniques: Derivative Assay, Immunoprecipitation, Residue, Activation Assay, Marker, Control

Journal: Stem cell research

Article Title: Src-family Tyrosine Kinase Activities are Essential for Differentiation of Human Embryonic Stem Cells

doi: 10.1016/j.scr.2014.09.007

Figure Lengend Snippet: Self-renewing hES cells (H1 and H7 lines) were cultured in the absence or presence of A-419259 (1.0 μM) for 16 h, followed by lysis and immunoprecipitation of c-Src and Lck. Each immunoprecipitate was then blotted with a phosphospecific antibody for the active form of each kinase (pY418 antibody, which recognizes the conserved phosphotyrosine residue in the activation loop of the kinase domain) as well as the respective SFK protein. Cell lysates were also blotted for pY418 as well as Oct4 as a marker of ES cell self-renewal. This experiment was repeated twice with comparable results; a representative example is shown.

Article Snippet: The SFK inhibitors SKI-1 and PP2 were purchased from EMD-Millipore-Calbiochem while A-419259 was purchased from Santa Cruz Biotechnology.

Techniques: Cell Culture, Lysis, Immunoprecipitation, Residue, Activation Assay, Marker

Journal: Stem cell research

Article Title: Src-family Tyrosine Kinase Activities are Essential for Differentiation of Human Embryonic Stem Cells

doi: 10.1016/j.scr.2014.09.007

Figure Lengend Snippet: The H7 line of hES cells was grown in mTeSR self-renewal medium or switched to differentiation medium in the absence (Control) or presence of A-419259 (1 μM). Cells were fixed, permeabilized and immunostained for the SFK Lck and the nuclear pluripotency factor Oct4 four days later. DAPI-stained nuclei are also shown. The merged image combines cells in the Lck and Oct4 panels only. This experiment was repeated three times with comparable results; a representative field of cells is shown for each condition.

Article Snippet: The SFK inhibitors SKI-1 and PP2 were purchased from EMD-Millipore-Calbiochem while A-419259 was purchased from Santa Cruz Biotechnology.

Techniques: Control, Staining

Journal: The Journal of Cell Biology

Article Title: Adhesion to fibronectin regulates Hippo signaling via the FAK–Src–PI3K pathway

doi: 10.1083/jcb.201501025

Figure Lengend Snippet: PI3K, PDK1, and Src regulation of nuclear YAP via Lats in serum-starved, subconfluent cells. (A) PI3K and PDK1 inhibitors relative to Lats. MCF-10A cells transfected with control, Nf2, or Lats1/2 siRNAs were serum starved and treated with DMSO (solvent control), 10 µM wortmannin (PI3K inhibitor), or 5 µM BX-795 (PDK1 inhibitor) for 30 min. On-target plus nontargeting pool was used as a control siRNA. Localization of endogenous YAP was identified by immunofluorescence staining. (B) SFK inhibitors and YAP localization. Serum-starved, low cell density MCF-10A cells were incubated with SFK inhibitors (10 µM each of PP2, dasatinib, SKI-1, and SU6656) for 30 min. 10 µM each of PP3 and imatinib were used as controls. YAP subcellular localization was determined by immunofluorescence staining. Alexa Fluor 594 secondary antibody was used for SU6656, which has high background green fluorescence. (C) Biochemical effects of Src inhibition. PP3- or PP2-treated MCF-10A cells were analyzed by Western blot using anti-YAP and anti–phospho-YAP (S127) antibodies. Phosphorylated YAP was detected by mobility shift on Phos-tag SDS-PAGE. (D) SFK inhibitors relative to Lats. MCF-10A cells transfected with control, Nf2, or Lats1/2 siRNA were serum starved and treated with 10 µM PP3 or PP2. After 30 min, cells were fixed for immunofluorescence staining with anti-YAP antibody. (E) Depletion of individual SFK. MCF-10A cells were transfected with control, Src, Fyn, or Yes siRNA. After serum starvation, subcellular localization of endogenous YAP was identified by immunofluorescence staining and quantified based on the criteria shown under the graph. More than 120 cells from four random views were quantified. (F) Src knockdown relative to Lats. MCF-10A cells were transfected with control, Src, Lats1/2, or combined siRNA of Src and Lats1/2. Cells were serum starved for 24 h before fixation and stained with anti-YAP antibody. (A, B, and D–F) One of three independent results is presented. Bars, 25 µm.

Article Snippet: The following inhibitors were used: SFK inhibitors SKI-1 and SU6656, Src/Abl inhibitor dasatinib monohydrate, Abl inhibitor imatinib mesylate, PDK1 inhibitor BX-795, FAK inhibitors PF-573228 and PF-562271 (Santa Cruz Biotechnology, Inc.), SFK inhibitor PP2 and its inactive analogue PP3 (EMD Millipore), and PI3K inhibitor wortmannin (Sigma-Aldrich).

Techniques: Transfection, Control, Solvent, Immunofluorescence, Staining, Incubation, Fluorescence, Inhibition, Western Blot, Mobility Shift, SDS Page, Knockdown