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

Article Title: Etk/BMX, a Btk family tyrosine kinase, and Mal contribute to the cross-talk between MyD88 and FAK pathways.

doi: 10.4049/jimmunol.180.5.3485

Figure Lengend Snippet: FIGURE 5. FAK is necessary for protein I/II and LPS-induced-Etk phosphorylation. FAK/ and FAK/ primary mouse embryo fibroblasts were incubated in either serum-free medium (T) or 5% FCS medium (C) or treated with LPS (1 g/ml) (A) or protein I/II (125 pM) (B) for the indi- cated times. Immunoprecipitation (Ip) was performed with anti- phosphotyrosine (anti-pY) mAbs (pY20), followed by immunoblotting (IB) with anti-Etk (clone 40) mouse mAbs. Lysates were also used for total protein blotting using anti-Etk (clone 40) mouse mAbs to confirm the pres- ence of equal levels of Etk protein between samples. The results shown are representative of three separate experiments.

Article Snippet: FAK / and FAK / primary mouse embryo fibroblasts (CRL-2644 and CRL-2645; American Type Culture Collection) were cultured as previously described (17).

Techniques: Phospho-proteomics, Incubation, Immunoprecipitation, Western Blot

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

Article Title: Etk/BMX, a Btk family tyrosine kinase, and Mal contribute to the cross-talk between MyD88 and FAK pathways.

doi: 10.4049/jimmunol.180.5.3485

Figure Lengend Snippet: FIGURE 6. Mal is phosphorylated via a FAK-dependent pathway. FAK/ and FAK/ primary mouse embryo fibroblasts were incubated in either serum-free medium (T) or 5% FCS medium (C) or stimulated with protein I/II (125 pM) (C) or LPS (1 g/ml) (A and B) for the indicated times. Immunoprecipitation (IP) was performed with anti-phosphotyrosine (anti-pY) mAbs (pY20) followed by immunoblotting with anti-Mal rabbit polyclonal Abs or anti-Mal rabbit polyclonal Abs followed by immunoblotting with anti- phosphotyrosine mAbs (pY20) (A). Lysates or immunoprecipitates were also used for total protein blotting using anti-Mal rabbit polyclonal Abs to confirm the presence of equal levels of Mal protein between samples. The results shown are representative of three separate experiments.

Article Snippet: FAK / and FAK / primary mouse embryo fibroblasts (CRL-2644 and CRL-2645; American Type Culture Collection) were cultured as previously described (17).

Techniques: Incubation, Immunoprecipitation, 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. 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: Biomolecules and Biomedicine

Article Title: KDM3A drives NSCLC proliferation and metastasis via H3K9 demethylation, EMT activation, and MMP-9 upregulation

doi: 10.17305/bb.2025.11251

Figure Lengend Snippet: Knockdown of KDM3A enhances H3K9me2 and modulates the cell invasion signaling pathway. (A) Two siRNAs inhibited KDM3A expression, resulting in an upregulation of H3K9me2 across the three cell lines, with no significant changes observed in KDM3B expression. The upper panel presents representative Western blot images, while the lower panel displays statistical analyses of the Western blot data ( n ═ 3). (B) The expression levels of E-cadherin, vimentin, MMP-9, pFAK, and FAK were evaluated using Western blotting. The upper panel shows representative Western blot images, and the lower panel provides statistical analyses ( n ═ 3). The ratio of pFAK to FAK was calculated after normalization to the β-actin loading control. (C) The expression levels of E-cadherin, vimentin, MMP-9, KDM3B, and KDM3C were assessed using qRT-PCR ( n ═ 3). All experiments were conducted a minimum of three times. Statistical analyses were performed using the Student's t -test, with significance levels indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001, ns: no significant differences. Abbreviations: KDM3A: Lysine-specific demethylase 3A; H3K9me2: Histone H3 lysine 9 dimethylation; KDM3B: Lysine-specific demethylase 3B; KDM3C: Lysine-specific demethylase 3C; siRNA: Small interfering RNA; FAK: Focal adhesion kinase; H1299/A549/HCC827: NSCLC cell lines; VIM: Vimentin.

Article Snippet: Membranes were then incubated overnight at 4 ∘ C with antibodies against KDM3A (GeneTex, 54313, 1:1000), KDM3B (Proteintech, 19915-1-AP, 1:1000), H3K9me2 (Cell Signaling Technology, 4658T, 1:2000), β-actin (Santa Cruz Biotechnology, sc-47778, 1:1000), matrix metalloproteinase-9 (MMP-9) (Santa Cruz Biotechnology, sc-393859, 1:1000), E-cadherin (Santa Cruz Biotechnology, sc-8426, 1:1000), Vimentin (Santa Cruz Biotechnology, sc-6260, 1:1000), FAK (Cell Signaling Technology, 3285S, 1:1000), pFAK (Tyr397) (Cell Signaling Technology, 3283, 1:1000), STAT3 (Cell Signaling Technology, 4904, 1:1000), pSTAT3 (Y705) (Cell Signaling Technology, 9131, 1:1000), and GAPDH (Proteintech, HRP-60004, 1:5000).

Techniques: Knockdown, Expressing, Western Blot, Control, Quantitative RT-PCR, Small Interfering RNA

Journal: Nature Communications

Article Title: The essential clathrin adapter protein complex-2 is tumor suppressive specifically in vivo

doi: 10.1038/s41467-025-57521-2

Figure Lengend Snippet: A Volcano plot of representing differential plasma membrane proteins comparing tumor samples from each cell type with and without AP2S1 (red – positive enrichment in absence of AP2, blue – negative enrichment, n = 3 technical replicates per condition; two-sided heteroscedastic t-test; fold-change > 2 and p < 0.05). B , C , D Venn diagrams representing overlapping GO terms from the indicated class. GO analysis was performed on proteins positively enriched at the cell membrane with the AP2S1 loss. Terms shared exclusively between PANC-1 and BXPC-3 cells are labeled and green highlighted terms are related to cell adhesion. E Immunoblot analysis of the indicated proteins from 10 tumors expressing sgAP2S1 and treated or not with doxycycline (Dox) before implantation to induce Cas9 (representative of n = 1 immunoblot of 5 independent tumors per condition). F Scatter plot superimposed on mean and standard deviation of AP2S1, plasma membrane ITGB1, and phosphorylated FAK from E ( n = 5 tumors per condition, two-sided t -test). G , I Representative fluorescent micrographs of internalized fluorescently labeled ITGB1 in ( G ) BXPC-3 and ( I ) CFPAC-1 cells. RFP correlates to the expression of the indicated shRNA . Red highlighted region indicates the image area quantified in H and J. Bar = 50 µm. H , J Scatter plot superimposed on mean and standard deviation of normalized ITGB1-488 fluorescence in RFP expressing cells quantified from G and I respectively ( H ) - n = 22, 29, 23, and 24 total images respectively over 2 independent experiments, J – n = 29, 33, 33, and 34 total images respectively over 2 independent experiments; one-way ANOVA). K Representative immunoblot of the indicated proteins from BXPC-3 cells cultured in 3D and expressing the indicated sgRNA and treated or not with doxycycline (Dox), Cilengitide, or FAK inhibitor (FAKi) (representative of n = 3 replicate immunoblots with inhibitor samples and 1 without over 4 independent experiments). L Scatter plot superimposed on mean and standard deviation of AP2M1 expression normalized to GAPDH from K ( n = 4, 4, 3, and 3 independent experiments respectively, one-way ANOVA). M Scatter plot superimposed on mean and standard deviation of pFAK expression normalized to total FAK from K ( n = 4, 4, 3, and 3 independent experiments respectively, one-way ANOVA). N ) Scatter plot superimposed on mean and standard deviation of % RFP cells after 7 days of growth, quantified from BXPC-3 competitive growth assays treated or not with doxycycline (Dox), Cilengitide, or FAK inhibitor (FAKi). ( n = 15 technical replicates per condition over 3 independent experiments; one-way ANOVA ). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Lysates were resolved by 18% SDS-PAGE, transferred to a PVDF membrane (Bio-Rad, 1704273), blocked in 5% milk or for phosphorylation specific antibodies 5% BSA, and immunoblotted by the following antibodies in 5% milk or 5% BSA (Sigma, A7906-500G): and ß-Tubulin (Sigma, T5201; diluted 1:5,000), PTEN (Cell Signaling, 9552; 1:1000), RPS16 (Abcam, ab26159; 1:2500), AP2S1(Abcam, ab128950; 1:250), AP2M1 (Abcam, ab75995; 1:250), GapDH(Santa Cruz, sc-365062; 1:5000), ITGB1 (Cell Signaling, 9699; 1:1000), FAK (Cell Signaling, 3285; 1:1000), pFAK (Cell Signaling, 8556; 1:1000), FTH1 (Cell Signaling, 4393; 1:500), TFRC (Cell Signaling, 13113; 1:500), IREB2 (Cell Signaling, 37135 s; 1:500), ITGA2 (Thermo Fisher Scientific, MA5-32306; 1:500), ITGA3 (Thermo Fisher Scientific, MA5-28565; 1:250), or ITGB6 (Cell Signaling, 95153; 1:1000).

Techniques: Clinical Proteomics, Membrane, Labeling, Western Blot, Expressing, Standard Deviation, shRNA, Fluorescence, Cell Culture

Journal: Cell Death and Differentiation

Article Title: LncRNA CASC9 promotes esophageal squamous cell carcinoma metastasis through upregulating LAMC2 expression by interacting with the CREB-binding protein

doi: 10.1038/s41418-018-0084-9

Figure Lengend Snippet: CASC9 activates FAK-PI3K/Akt signaling pathways through LAMC2. a Western blot analysis showed altered levels of pFAK, pPI3K, and pAkt after CASC9 or LAMC2 knockdown in KYSE150 and KYSE450 cells. b Western blot analysis of FAK-PI3K/Akt signaling pathways of the rescue experiment. c RT-qPCR analysis of MMP10 and MMP13 expression. d , e The expression of MMP10 and MMP13 in KYSE150 and KYSE450 cells after CASC9 knockdown. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: An anti-GAPDH antibody (#KC-5G5, KangChen, China) was used as an internal reference; anti-Laminin-5 (γ2 chain) (MAB19562) was from Millipore (Millipore, USA); anti-histone H3 (D2B12), anti-ITGB4 (#4707), FAK Antibody Sampler Kit (#9330), anti-PI3K Kinase p110α (#4249), anti-Phospho-PI3 Kinase p85 (Tyr458)/p55(Tyr199) (#4228), anti-AKT (pan) (#4691), and anti-phospho-Akt (Ser473) (#4060) were from Cell Signaling Technology (CST, USA); anti-MMP10 antibody (ab199688) and anti-MMP13 (ab39021), antibody anti-KAT3A/CBP (ab2832), and anti-histone H3 (acetyl K27) (ab4729) were from Abcam (Abcam, UK).

Techniques: Protein-Protein interactions, Western Blot, Knockdown, Quantitative RT-PCR, Expressing

Journal: Cell Death and Differentiation

Article Title: LncRNA CASC9 promotes esophageal squamous cell carcinoma metastasis through upregulating LAMC2 expression by interacting with the CREB-binding protein

doi: 10.1038/s41418-018-0084-9

Figure Lengend Snippet: Model of CASC9 interaction with CBP and the signaling pathways involved in pro-metastatic function. a In the nucleus, CASC9 recruits CBP and modulates H3K27ac levels at the LAMC2 promoter, thereby facilitating gene transcription. b In the cytoplasm, LAMC2 is exported to the extracellular matrix and interacts with ITGB4 to activate FAK-PI3K/Akt signaling pathways, thereby promoting ESCC metastasis

Article Snippet: An anti-GAPDH antibody (#KC-5G5, KangChen, China) was used as an internal reference; anti-Laminin-5 (γ2 chain) (MAB19562) was from Millipore (Millipore, USA); anti-histone H3 (D2B12), anti-ITGB4 (#4707), FAK Antibody Sampler Kit (#9330), anti-PI3K Kinase p110α (#4249), anti-Phospho-PI3 Kinase p85 (Tyr458)/p55(Tyr199) (#4228), anti-AKT (pan) (#4691), and anti-phospho-Akt (Ser473) (#4060) were from Cell Signaling Technology (CST, USA); anti-MMP10 antibody (ab199688) and anti-MMP13 (ab39021), antibody anti-KAT3A/CBP (ab2832), and anti-histone H3 (acetyl K27) (ab4729) were from Abcam (Abcam, UK).

Techniques: Protein-Protein interactions