Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Inhibition of cell-matrix adhesions prevents cartilage chondrocyte death following impact injury.

doi: 10.1002/jor.22523

Figure Lengend Snippet: Figure 3. Confocal microscopy and cell viability immediately after impact injury. (A) Confocal micrographs show live (green) and dead (red) chondrocytes in an impact site in an un-treated control explant, and in explants treated with 10 mM SFKi and either 10 or 100 mM FAKi. Compared to control, fewer dead chondrocytes were observed in SFKs or FAKi treated groups. (B) Statistical analysis revealed that chondrocyte viability was significantly higher in SFKs or FAKi treated explants compared to control. Between two tested concentrations, 100 mM FAKi was more effective than 10 mM. Asterisk represents statistically significant (p < 0.05, p < 0.01). Bars ¼ 500 mm.

Article Snippet: Published by Wiley Periodicals, Inc. 448 JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2014 After 2 days, the explants were randomly distributed and were treated with fresh culture medium containing 10 or 100mM focal adhesion kinase inhibitor (FAKi) (Santa Cruz Biotechnology, Dallas, TX) to block phosphorylation of FAK at the kinase domain (Try 397) or were treated with fresh culture medium containing 10mM Src family kinase inhibitor (SFKi) (Selleckchem, Houston, TX) to block phosphorylation of SFKs at kinase domain (Tyr 416) for 2h.

Techniques: Confocal Microscopy, Control

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Inhibition of cell-matrix adhesions prevents cartilage chondrocyte death following impact injury.

doi: 10.1002/jor.22523

Figure Lengend Snippet: Figure 5. Kinetics of FAK and SFKi by western blot analysis. (A) Immunoblot analysis showed that treatment with 10 ng/ml IL-1b and 100 ng/ml TNF-a for 30 min significantly increased SFKs phosphorylation at Tyr 416. 0.1, 1, and 10 mM SFKi diminished this response dose dependently. (B) Analysis of the integrated densities of the bands with the phosphor- to total SFKs ratio. C represents untreated control and Cþ represents 10 ng/ml IL-1b and 100 ng/ml TNF-a treated only. (C) Immunoblots show that treatment with 100 nM fMLF for 30 min did not enhance FAK phosphorylation at Tyr 397; however 100 mM FAKi significantly reduced FAK phosphorylation among tested concentrations of 1, 10, and 100 mM. (D) Analysis of the integrated densities of the bands with the phosphor- to total FAK ratio. C represents untreated control and Cþ represents 100 nM fMLF treated only.

Article Snippet: Published by Wiley Periodicals, Inc. 448 JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2014 After 2 days, the explants were randomly distributed and were treated with fresh culture medium containing 10 or 100mM focal adhesion kinase inhibitor (FAKi) (Santa Cruz Biotechnology, Dallas, TX) to block phosphorylation of FAK at the kinase domain (Try 397) or were treated with fresh culture medium containing 10mM Src family kinase inhibitor (SFKi) (Selleckchem, Houston, TX) to block phosphorylation of SFKs at kinase domain (Tyr 416) for 2h.

Techniques: Western Blot, Phospho-proteomics, Control

Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCa depletion significantly inhibits spreading-induced signaling. (A) siRNA-transfected cells held in suspension for 1 h (left panel) or plated on collagen IV-coated dishes for the indicated times (minutes, right panel) were lysed and subjected to western blotting with anti-active FAK [pFAK(Y397)], active Src [pSrc(Y416)] and anti-phosphorylated paxillin [pPax(Y118)] antibodies. (B) Protein phosphorylation in control cells at 30 min after plating was set as 100%. The results represent a summary from five to seven experiments. *P<0.05; **P<0.01. (C) Lysates from plated cells treated as in A were analyzed by western blotting with antibodies against Src-dependent FAK phosphorylation sites (Y576 and Y925). The blots shown represent one of three independent experiments.

Article Snippet: Antibodies against the following proteins were used: CLCa (1:1000, sc-28276), CLCb (1:500, sc-376414), actin (1:1000, sc-1616) from Santa Cruz Biotechnology, FAK (1:2000, 610088) and β1-integrin (1:1000, 610467) from BD Transduction Labs, phosphorylated FAK(Y397) (1:1000, 44-624G), phosphorylated paxillin(Y118) (1:1000, 44-722G) from Fisher Scientific, Src (1:2000, 2108), phosphorylated Src(Y416) (1:1000, MAB2685, 2101), phosphorylated FAK(Y576) (1:1000, 3281), FAK(Y925) (1:1000, 3284) from Cell Signaling, phosphorylated Src(Y416) (1:1000, MAB2685) from RD Systems, WAVE1/Scar (1:1000, 07-037), Rac1 (1:2000, 05-389) from Millipore.

Techniques: Transfection, Western Blot

Journal: bioRxiv

Article Title: The c-Src inhibitor eCF506 diminishes opioid tolerance creating bias against β-arrestin2 recruitment

doi: 10.1101/2025.07.01.662507

Figure Lengend Snippet: Concentration-response relationships of DAMGO as a recruiter of β-arrestin2 to μ receptors in PathHunter CHO cells transiently overexpressing ( A ) full-length (residues 1-536; WT) versus C-terminally truncated (residues 1-249) human c-Src and ( B ) functional (residues 250-536) versus catalytically non-functional (residues 250-536(K298M)) N-terminally truncated human c-Src. The insert in (A) demonstrates overexpression of C-terminally truncated c-Src using an antibody against HA (1:1000). Overexpression of other c-Src mutants is depicted in Supplementary Figure 5A. WT responses in (B) are depicted as a dashed line. Data are expressed as a percentage of maximum luminescence produced by DAMGO in cells overexpressing WT c-Src (% WT). Data from individual replicates were plotted and fitted with logistics functions to derive efficacy (E MAX ) and potency (EC 50 ) parameters, presented in . Data in A and B are the mean ± SEM of 9 replicates.

Article Snippet: All constructs are based on human c-Src WT (1-536) c-Src (plasmid #42202; RRID: Addgene_42202), c-terminus truncated (1-249) c-Src (plasmid #42204; RRID: Addgene_42204), n-terminus truncated (250-536) c-Src (plasmid #42208; RRID: Addgene_42208), and kinase dead (K298M) c-Src (plasmid #42210; RRID: Addgene_42210), were donated by Dr Robert Lefkowitz.

Techniques: Concentration Assay, Functional Assay, Over Expression, Produced

Journal: bioRxiv

Article Title: The c-Src inhibitor eCF506 diminishes opioid tolerance creating bias against β-arrestin2 recruitment

doi: 10.1101/2025.07.01.662507

Figure Lengend Snippet: Concentration-response relationships of DAMGO to recruit β-arrestin2 to human μ receptors in PathHunter CHO cells transiently overexpressing ( A ) full-length (residues 1-536; WT), ( B ) C-terminally truncated (residues 1-249) or ( C ) N-terminally truncated and catalytically inactive (residues 250-536(K298M)) human c-Src following 16-h exposure to PP2, PP3, eCF506 (300 nM) or an equal volume of DMSO. Data are expressed as a percentage of maximum luminescence (% Max RLU) produced by DAMGO in DMSO exposed cells present in duplicate on each plate i.e., cells overexpressing (A) WT, (B) Src 1-249 or (C) Src 250-536(K298M) are each expressed relative to the maximum β-arrestin2 recruitment of each construct caused by DAMGO. Data from individual replicates were plotted and fitted with logistics functions to derive efficacy (E MAX ) and potency (EC 50 ) parameters, presented in Supplementary Table 3. Data are the mean ± SEM of 5-6 replicates.

Article Snippet: All constructs are based on human c-Src WT (1-536) c-Src (plasmid #42202; RRID: Addgene_42202), c-terminus truncated (1-249) c-Src (plasmid #42204; RRID: Addgene_42204), n-terminus truncated (250-536) c-Src (plasmid #42208; RRID: Addgene_42208), and kinase dead (K298M) c-Src (plasmid #42210; RRID: Addgene_42210), were donated by Dr Robert Lefkowitz.

Techniques: Concentration Assay, Produced, Construct