Journal: Experimental and molecular pathology

Article Title: Focal adhesion kinase coordinates costamere-related JNK signaling with muscle fiber transformation after Achilles tenotomy and tendon reconstruction.

doi: 10.1016/j.yexmp.2019.03.006

Figure Lengend Snippet: Fig. 9. Effects of FRNK-mediated inhibition of FAK on hypertrophy signaling. Box Whisker plots of the (specific) phosphorylation of Y397-FAK (A), S2448-mTOR (B), T183/Y185-JNK (C) and S63-cJUN (D) in not tenotomized soleus muscle 7 days after co-transfection of muscle pairs with pCMV & pCMV-FAK and pCMV-FRNK & pCMV-FAK. Circles indicate outliers. + and ++, p < .05 and p < .01 vs. pCMV & pCMV-FAK (repeated-measures ANOVA). Values represented normalized intensities respective to the pCMV & pCMV-FAK transfected muscles. E) Example western blot showing the detection of FAK and FRNK (top) in 10 μg protein homogenate from a soleus muscle pair being co-transfected with pCMV & pCMV-FAK and pCMV-FRNK & pCMV-FAK, respectively. Below, respective actin band.

Article Snippet: An antibody directed against the Nterminal region of FAK (AF4467, R&D systems) and an antibody that detects the phosphorylation at tyrosine 397 (P00151, Boster Biological Technology) were biotinylated (Biotin-NH2 labeling Kit, KT-221, Kamiya Biomedical Company) and used as capture antibodies.

Techniques: Inhibition, Whisker Assay, Phospho-proteomics, Cotransfection, Transfection, Muscles, Western Blot

Journal: Experimental and molecular pathology

Article Title: Focal adhesion kinase coordinates costamere-related JNK signaling with muscle fiber transformation after Achilles tenotomy and tendon reconstruction.

doi: 10.1016/j.yexmp.2019.03.006

Figure Lengend Snippet: Fig. 10. Inter-relationships. Hierarchical networks of the correlations between measured molecular and cellular parameters in all assessed soleus mus- cles. Correlated parameters (nodes) are connected though lines (edges; solid dark-grey line: positive correlation, back- ward slashed grey line: negative correlation). Abbreviations: body_m_e, body mass at the end of the experiment; body_m_s, body mass at the start of the experiment; %con_tis, percentage area of connective tissue; %f-fast, percentage of fast (II) type muscle fibers; %f-hybrid, percentage of hybrid (I/II) type muscle fibers; %f-inuc, percentage of fibers with internal nu- clei; %f-slow, percentage of slow (I) type muscle fibers; FAK, levels of FAK; FRNK, levels of FRNK; g-VN, gamma-vinculin; GM_mass, mass of gastrocnemius muscle; MCSA, mean cross sectional area of muscle fibers; m-VN, meta-vinculin; s-pFAK, specific phosphorylation of Y397-FAK; s-pJNK, specific phosphorylation of T183/Y185-JNK; s-pmTOR, specific phosphorylation of S2448-mTOR; s-pP70S6K, specific phos- phorylation of T421/S424-P70S6K; SOL_mass, mass of soleus muscle.

Article Snippet: An antibody directed against the Nterminal region of FAK (AF4467, R&D systems) and an antibody that detects the phosphorylation at tyrosine 397 (P00151, Boster Biological Technology) were biotinylated (Biotin-NH2 labeling Kit, KT-221, Kamiya Biomedical Company) and used as capture antibodies.

Techniques: Phospho-proteomics

Journal: The Journal of Cell Biology

Article Title: Src activation by Chk1 promotes actin patch formation and prevents chromatin bridge breakage in cytokinesis

doi: 10.1083/jcb.201802102

Figure Lengend Snippet: Src localizes to actin patches in cells with chromatin bridges. (A–E) Localization of Src (A), phosphorylated Src-Y419 (pSrc-Y419; B), phosphorylated FAK-Y925 (pFAK-Y925; C and D), or cortactin (E). Cells were transfected with negative siRNA (control) or siChk1, or treated with PP2 for 5 h. (F) Localization of GFP-Chk1. Broken DNA bridges are indicated by dotted arrows, and the bases of the intercellular canals are indicated by solid arrows. Images are representative of 20 cells from two independent experiments. Insets show 1.6× magnification of the canals bases. Bars, 5 µm.

Article Snippet: Mouse monoclonal antibodies against α-tubulin (DM1A) and actin (AC-40) were from Sigma-Aldrich, mouse monoclonal anti–lamin B2 (E-3) was from Thermo Fisher Scientific (33-2100), and rabbit polyclonal antibody against phospho–FAK-Y925 (AF44671) was from R&D Systems.

Techniques: Transfection, Control

Journal: The Journal of Cell Biology

Article Title: Src activation by Chk1 promotes actin patch formation and prevents chromatin bridge breakage in cytokinesis

doi: 10.1083/jcb.201802102

Figure Lengend Snippet: Chk1-inhibition reduces Src kinase activity. (A and B) Cells transfected with negative siRNA (control) or siChk1 were trypsinized and seeded on fibronectin-coated dishes in the absence or presence of PP2 for 1 h. Western blot analysis of total phospho–Src-Y419 (pSrc-Y419), Src, phospho–FAK-Y925 (pFAK-Y925), FAK, or actin. NS, nonspecific. Relative band intensity values are shown, and values in control were set to 1. (C) Chk1 depletion reduces cell spreading. Cells were seeded on fibronectin-coated dishes and phase-contrast images taken at various times after plating. Bars, 50 µm. (D) Percentage of spread cells (i.e., flattened and not highly reflective) from C. Error bars show the SD from the mean from three independent experiments. Approximately 750–1,000 cells were analyzed per experiment. (E) Cells were seeded on fibronectin-coated dishes and analyzed by wound healing assay. Phase-contrast images at 0 h and 12 h after wounding are shown. Bars, 200 µm. (F) Wound area covered calculated from cells as in E. Error bars show the SD from the mean from four independent experiments. **, P < 0.01; ***, P < 0.001 compared with the control. Statistically significant differences were determined by ANOVA and Student’s t test.

Article Snippet: Mouse monoclonal antibodies against α-tubulin (DM1A) and actin (AC-40) were from Sigma-Aldrich, mouse monoclonal anti–lamin B2 (E-3) was from Thermo Fisher Scientific (33-2100), and rabbit polyclonal antibody against phospho–FAK-Y925 (AF44671) was from R&D Systems.

Techniques: Inhibition, Activity Assay, Transfection, Control, Western Blot, Wound Healing Assay

Journal: The Journal of Cell Biology

Article Title: Src activation by Chk1 promotes actin patch formation and prevents chromatin bridge breakage in cytokinesis

doi: 10.1083/jcb.201802102

Figure Lengend Snippet: Mutation of human Src-S51 to alanine reduces Src catalytic activity. (A) Chk1 kinase assay. Top: Autoradiography analysis ( 32 P) of phosphorylated Src using purified WT or S51A GST-Src (1–256) substrate. Bottom: Western blot (WB) analysis of total GST. Top and bottom panels are from the same gel. (B) Alignment of Src protein sequences. Human serine 51 is marked by an asterisk. (C) Chk1 in vitro kinase assay. Western blot analysis of phosphorylated (pSrc-S51) by using the anti-pS51 antiserum along with total GST-Src. (D) In vitro kinase assay using recombinant GST-Chk1, His-Src, and purified GST-FAK (871–1020) as substrate. Western blot analysis of phosphorylated FAK-Y925 (pFAK-Y925) and pSrc-S51 using phosphospecific antibodies, and Ponceau staining of GST-FAK (871–1020) and His-Src. Relative band intensity values are shown, and values in the second lane from left (His-Src) were set to 1. (E) Immunoprecipitation (IP) kinase assay using GST-FAK (871–1020) as substrate. Top: Western blot analysis of GFP-associated FAK-Y925 phosphorylation and immunoprecipitated GFP, and Ponceau staining of GST-FAK (871–1020). Bottom: Western blot analysis of total GFP and actin. Relative band intensity values are shown, and values in GFP–Src-WT were set to 1. Ab, antibody. (F) Immunoprecipitation-kinase assay in the absence or presence of siChk1. Top: Western blot analysis of GFP-associated FAK-Y925 phosphorylation, immunoprecipitated GFP, and GST-FAK (871–1020). Bottom: Western blot analysis of total GFP and actin. Relative band intensity values are shown, and values in GFP–Src-WT without siChk1 were set to 1.

Article Snippet: Mouse monoclonal antibodies against α-tubulin (DM1A) and actin (AC-40) were from Sigma-Aldrich, mouse monoclonal anti–lamin B2 (E-3) was from Thermo Fisher Scientific (33-2100), and rabbit polyclonal antibody against phospho–FAK-Y925 (AF44671) was from R&D Systems.

Techniques: Mutagenesis, Activity Assay, Kinase Assay, Autoradiography, Purification, Western Blot, In Vitro, Recombinant, Staining, Immunoprecipitation, IP-Kinase Assay, Phospho-proteomics

Journal: The Journal of Cell Biology

Article Title: Src activation by Chk1 promotes actin patch formation and prevents chromatin bridge breakage in cytokinesis

doi: 10.1083/jcb.201802102

Figure Lengend Snippet: Chk1 depletion reduces Src-S51 phosphorylation. (A) Immunoprecipitation (IP) kinase assay using GST-FAK (871–1020) as substrate. Top: Western blot analysis of GFP-associated FAK-Y925 phosphorylation (pFAK-Y925) and immunoprecipitated GFP as well as Ponceau staining of GST-FAK (871–1020). Bottom: Western blot analysis of total GFP and actin. Relative band intensity values are shown, and values in GFP–Src-WT were set to 1. (B) Localization of phosphorylated Src-S51 (pSrc-S51). Cells were transfected with negative siRNA (control), siChk1, or siSrc. Broken DNA bridges are indicated by dotted arrows, and the bases of the intercellular canals are indicated by solid arrows. Insets show 1.6× magnification of the canals bases. Images are representative of 20 cells from two independent experiments. (C) Cells transfected as in B were seeded on fibronectin-coated slides for 1 h. Insets show 2× magnification of membrane ruffles. Bars, 5 µm. (D) Phosphorylated Src-S51 at membrane ruffles. Relative green fluorescence intensity from C is shown, and values in control were set to 1. Error bars show the SD from the mean. n = 20 cells from two independent experiments. ***, P < 0.001 compared with the control. Statistically significant differences were determined by ANOVA and Student’s t test. (E) Coimmunoprecipitation from asynchronous cells. Chk1 or Src were detected by Western blotting. Ab, antibody.

Article Snippet: Mouse monoclonal antibodies against α-tubulin (DM1A) and actin (AC-40) were from Sigma-Aldrich, mouse monoclonal anti–lamin B2 (E-3) was from Thermo Fisher Scientific (33-2100), and rabbit polyclonal antibody against phospho–FAK-Y925 (AF44671) was from R&D Systems.

Techniques: Phospho-proteomics, Immunoprecipitation, IP-Kinase Assay, Western Blot, Staining, Transfection, Control, Membrane, Fluorescence