phosphorylated p fak  (Cell Signaling Technology Inc)

Journal: Molecular Medicine Reports

Article Title: Fibrosis factor CTGF facilitates VCAM‑1‑dependent monocyte adhesion to osteoarthritis synovial fibroblasts via the FAK and JNK pathways

doi: 10.3892/mmr.2025.13489

Figure Lengend Snippet: FAK activation is involved in CTGF-induced VCAM-1 synthesis and monocyte adhesion. (A and B) VCAM-1 expression (n=3) and (C) THP-1 adhesion (n=4) in OASFs incubated with a FAK inhibitor (10 µM) and then stimulated with CTGF for 24 h. The densitometry analysis of (B) was quantified (n=3; right panels). (D) Western blot analysis showing FAK phosphorylation in OASFs treated with CTGF. The densitometry analysis of (D) was quantified (n=3; lower panels). *P<0.05 vs. control group. # P<0.05 vs. CTGF-treated group. FAK, focal adhesion kinase; CTGF, connective tissue growth factor; VCAM-1, vascular cell adhesion molecule-1; p-, phosphorylated.

Article Snippet: CTGF recombinant protein was obtained from PeproTech, Inc. Antibodies against phosphorylated (p-)FAK (cat. no. 3283S) and p-JNK (cat. no. 9251S) were acquired from Cell Signaling Technology, Inc. Antibodies against VCAM-1 (cat. no. GTX110684), FAK (cat. no. GTX50489), JNK (cat. no. GTX52360) and β-actin (cat. no. GTX109639) were acquired from GeneTex International Corporation.

Techniques: Activation Assay, Expressing, Incubation, Western Blot, Phospho-proteomics, Control

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A. Data from mass spectrometry of HEK-293T cells demonstrating Bclaf1 on Flag-FERM-PTK2 immunoprecipitate. B. Interaction networks of PTK2 and Bclaf1 on HEK293T obtained from IntAct ( https://www.ebi.ac.uk/intact/home ), including association and physical association. C-D . Bclaf1 immunoblots of PTK2 immunoprecipitated from control H9c2 extracts confirming the PTK2-Bclaf1 interaction, and vice versa. PTK2 (C) and Bclaf1 (D) served as loading control. E. Super-resolution images of H9c2 cells on basal conditions, stained for PTK2, Bclaf1, actin, and nucleus. Scale bar = 10 μm. Nucleus zoomed image scale bar = 5 μm. F-G. Z-stack slices of nuclear planes showing the association of PTK2 with Bclaf1 (arrows). Scale bar = 1 μm. Zoomed images scale bar = 0.1μm. H. Proximity Ligation Assay (PLA) demonstrating the interaction between PTK2 and Bclaf1 in untreated cells. Scale bar: 5 μm. Green: PTK2; Red: Bclaf1; Blue: nucleus; Grey: actin; magenta: PLA.

Article Snippet: Other combinations were performed with antibodies against phosphorylated PTK2 (Thermo Fisher, 44-625G – 1:200) and Hsp70 (Thermo Fisher, MA3-006 – 1:50).

Techniques: Mass Spectrometry, Western Blot, Immunoprecipitation, Control, Staining, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A. Triple-label SR-SIM image showing PTK2 and Bclaf1 concentrated on biomolecular condensates on the nucleus of H9c2 cardiomyocytes treated with dox. Scale bar = 10 µm. 1. Fluorescence line profile plot showing PTK2 colocalizing with ubiquitin outside the Bclaf1 condensate. Scale bar = 0.5 µm. 2. Fluorescence line profile plot showing low ubiquitin level inside the Bclaf1 condensate, where PTK2 is enriched. Scale bar = 0.5 µm B. Pearson correlation coefficient between Bclaf1 and PTK2, ubiquitin and PTK2, and ubiquitin and Bclaf1. C. PTK2 immunoblots of extracts from control (CT) and dox-treated (DOX) H9c2 cells and bar graph showing a decrease in PTK2 levels after dox treatment. D. RT-qPCR assay shows no difference in PTK2 mRNA levels in cardiomyocytes on basal conditions or after dox treatment. GAPDH was used as endogenous control. E. Western-blot of PTK2 and ubiquitin comparing soluble (S) and insoluble (P) fractions of H9c2 cardiomyocytes. F. Super-resolution images of PTK2, Bclaf1, and ubiquitin staining after 36 hours of recovery from dox treatment. Scale bar = 5 µm. 1. Fluorescence line profile plot showing low PTK2 ubiquitination inside Bclaf1 condensate. Scale bar = 0.5 µm. 2. Fluorescence line profile plot showing PTK2 ubiquitinated and agglomerated outside Bclaf1 condensate. G. Graphical representation of PTK2-Bclaf1 undergoing LLPT to avoid PTK2 ubiquitination and aggregation. H. Western-blot showing ubiquitin accumulation on cardiomyocytes treated with dox and co-treated with dox and MG132. I. SR-SIM image showing the accumulation of ubiquitinated PTK2 on the nucleus of cardiomyocytes co-treated with dox and MG132. Scale bar of nuclear image = 5 µm. (1) Orthogonal Z projection showing yz planes and xz planes of a PTK2 agglomerate, which colocalize with ubiquitin. Scale bar = 0.5 µm. J. Cell number count after dox combined with MG132 treatment, as indicated. Green: PTK2; Red: Bclaf1; Grey: Ubiquitin. Asterisks demonstrates statistical significance, while ns means P > 0.05; * means P ≤ 0.05; ** means P ≤ 0.01; *** means P ≤ 0.001 and **** means P ≤ 0.0001.

Article Snippet: Other combinations were performed with antibodies against phosphorylated PTK2 (Thermo Fisher, 44-625G – 1:200) and Hsp70 (Thermo Fisher, MA3-006 – 1:50).

Techniques: Fluorescence, Ubiquitin Proteomics, Western Blot, Control, Quantitative RT-PCR, Staining

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A-B. Immunofluorescence images of H9c2 cardiomyocytes showing PTK2 and Bclaf1 reorganization in dot-like structures after dox treatment, respectively. C. Immunofluorescence images of pY397-PTK2 (pPTK2) showing nuclear PTK2 activation after dox treatment. D. Bar graphs show the nuclear fluorescence intensity of PTK2, Bclaf1, and pPTK2 on basal conditions and after dox treatment. Asterisks means statistical significance, while ns means P > 0.05; * means P ≤ 0.05; ** means P ≤ 0.01; *** means P ≤ 0.001 and **** means P ≤ 0.0001. E-F. pY397-PTK2, PTK2, GAPDH, and Histone H1 (H1) specific immunoblots of cytoplasmic and nuclear extract of H9c2 control or treated with dox. Dox treatment: 1 µM; 12h. Green: PTK2 and pPTK2; Grey: Actin; Red: Bclaf1; Blue: nucleus. Scale bar = 5 µm. Scale bar of zoomed images = 3 µm.

Article Snippet: Other combinations were performed with antibodies against phosphorylated PTK2 (Thermo Fisher, 44-625G – 1:200) and Hsp70 (Thermo Fisher, MA3-006 – 1:50).

Techniques: Immunofluorescence, Activation Assay, Fluorescence, Western Blot, Control

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A-B . Bclaf1 immunoblots of PTK2 immunoprecipitate from dox-treated H9c2 cardiomyocytes extracts, and vice versa. PTK2 (A) and Bclaf1 (B) served as input loading control. C. Immunofluorescence images of dox-treated cardiomyocytes showing the rearrangement of Bclaf1 into nuclear condensates containing PTK2. Below, Z-stack from nuclear planes, demonstrating the association of PTK2 and Bclaf1 in different planes. Scale bar of upper panels = 5 µm, 1 µm, 2 µm, and 0.5 µm, respectively. Scale bar of Z-stack images = 0.5 µm. D. PTK2-Bclaf1 condensate image showing strong colocalization in contrast with a no-complex region. Arrowhead shows the 3 PTK2 bright peaks. Scale bar = 0.5 µm. E. Orthogonal Z projection showing yz planes, xz planes and 3D reconstruction of the Bclaf1 biomolecular condensate containing PTK2 shown in “d”. F. Fluorescence line profile plot of the yellow horizontal line in (E) showing PTK2 inside the Bclaf1 condensate. Green: PTK2; Red: Bclaf1; Blue: nucleus. Dox treatment: 1 µM; 12h. G. Proximity Ligation Assay showing increased interaction between PTK2 and Bclaf1 after dox treatment. Scale bar = 5 µm.

Article Snippet: Other combinations were performed with antibodies against phosphorylated PTK2 (Thermo Fisher, 44-625G – 1:200) and Hsp70 (Thermo Fisher, MA3-006 – 1:50).

Techniques: Western Blot, Control, Immunofluorescence, Fluorescence, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A. The structure of Bclaf1, an intrinsically disordered protein (IDP), with an inner core of 4 alpha helices and flexible long loops was obtained using AlphaFold3. PAE matrix indicates low error values only on the diagonal, reflecting the Bclaf1 unfolding nature. B. PTK2 structure, with three main domains and a long intrinsically disordered region (IDR) linking the Kinase and FAT domains obtained by AlphaFold3 platform is shown. PAE matrix shows the predicted error in Angstroms. C. Predicted PTK2-Bclaf1 modeled using AlphaFold3 docking tool and PAE matrix, indicating low error values for the PTK2 domains and a higher error value for the IDR. Interfaces of PTK2-Bclaf1 interaction with intermediate error values are shown. D. LLPS propensity scores for Bclaf1, PTK2, IDR PTK2-N-terminal, Q686 – S923 IDR PTK2 sequence, and the PTK2-Bclaf1 complex obtained using FuzDrop and PSPredictor. E. Residue-based droplet promoting probability of PTK2 and Bclaf1 obtained by FuzDrop.

Article Snippet: Other combinations were performed with antibodies against phosphorylated PTK2 (Thermo Fisher, 44-625G – 1:200) and Hsp70 (Thermo Fisher, MA3-006 – 1:50).

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A. FRAP fitted graphs of condensate and out of condensate nuclear regions of H9c2 cells transfected with EGFP-Bclaf1 and EGFP-PTK2, in individual experiments. Bclaf1 and PTK2 present higher diffusion on condensate regions in relation to non-condensate areas. Images show the fluorescence and differential interference contrast (DIC) images of EGFP-Bclaf1 and EGFP-PTK2 expressing cells. Scale bar = 5 μm. B. Mobile fraction and constant of the fluorescence recovery rate for Bclaf1 and PTK2. C. Lifetime images of H9c2 cells expressing GFP-Bclaf1 or GFP-PTK2 fluorescent proteins. D. Bar graph of EGFP-Bclaf1 and EGFP-PTK2 fluorescence lifetime in condensates and no condensate areas. Asterisks means statistical significance, while ns means P > 0.05; * means P ≤ 0.05; ** means P ≤ 0.01; *** means P ≤ 0.001 and **** means P ≤ 0.0001.

Article Snippet: Other combinations were performed with antibodies against phosphorylated PTK2 (Thermo Fisher, 44-625G – 1:200) and Hsp70 (Thermo Fisher, MA3-006 – 1:50).

Techniques: Transfection, Diffusion-based Assay, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A. Super-resolution images of H9c2 cardiomyocytes showing the colocalization of Hsp70 (gray) with PTK2 (green) and Bclaf1 (red) on the condensate region. Scale bar = 10 µm. Scale bar of zoomed images = 0.5 µm. B. FRAP graph showing the fluorescence recovery after photobleaching of EGFP-Bclaf1 and EGFP-PTK2 after Hsp70 inhibition (iHsp70). Asterisks demonstrates statistical significance, while ns means P > 0.05; * means P ≤ 0.05; ** means P ≤ 0.01; *** means P ≤ 0.001 and **** means P ≤ 0.0001. C. Time-course experiments using CellROX show an increasing oxidative stress during dox treatment. Scale bar = 5 µm. D. Bar Graph shows the percentage of cells with Bclaf1 clusters from control (CT) to 12h of dox treatment, as indicated (n=80 for CT, n=59 for 1h, n=94 for 3h, n=76 for 6h, and n=91 for 12h). E. Bar graph shows the MTT absorbance at 540 nm. F. Western Blot images of p53, p53 phosphorylated at Ser 15 (p-p53-S15), and cleavage caspase-3. α-Tubulin served as loading control. Bar Graphs are shown in Supplementary Figure 4.

Article Snippet: Other combinations were performed with antibodies against phosphorylated PTK2 (Thermo Fisher, 44-625G – 1:200) and Hsp70 (Thermo Fisher, MA3-006 – 1:50).

Techniques: Fluorescence, Inhibition, Control, Western Blot

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A. MS/MS spectrum showing the PTK2 identified peptide (m/z 561.3040) with the chemical modification (K-GG) characteristic of ubiquitination. B. Linear representation of rat and human PTK2 demonstrating their domains and the PTK2 ubiquitination site on rat, lysine 926, and in human, lysine 923. C. Cartoon representation of PTK2 showing the K926 ubiquitination site on the FAT domain. D. Hydrostatic representation of PTK2-Bclaf1 interaction complex, showing K926 site being sterically occluded by Bclaf1. The FERM domain is represented in blue, the Kinase in red, and the FAT domain in yellow.

Article Snippet: Other combinations were performed with antibodies against phosphorylated PTK2 (Thermo Fisher, 44-625G – 1:200) and Hsp70 (Thermo Fisher, MA3-006 – 1:50).

Techniques: Tandem Mass Spectroscopy, Modification, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Bclaf1 biomolecular condensates protect nuclear PTK2 from ubiquitin-proteasome system promoting cardiomyocyte survival during oxidative stress

doi: 10.1101/2025.02.04.636487

Figure Lengend Snippet: A. Western blot image and bar graph confirming the knockdown of Bclaf1 (KD-Bclaf1) in relation to control (CT) H9c2 cardiomyocytes. B. Western blotting images show the increased ubiquitination after Bclaf1 knockdown in relation to control cardiomyocytes, both treated with dox. C. SR-SIM image and bar graph show the ubiquitin accumulation on the nucleus of KD-Bclaf1 cardiomyocytes treated with dox. Scale bar = 5 µm. D. SR-SIM image shows the effect of Bclaf1 knockdown on disrupting condensates assembly. Scale bar = 5 µm. E. SR-SIM image and line profile plot showing Bclaf1 knockdown increases PTK2 agglomeration and ubiquitination on the nucleus. Scale bar = 5 µm. Scale Bar of zoomed image = 0.5 µm. 1. Fluorescence line profile plot showing low PTK2 ubiquitination in PTK2 condensate. 2. Fluorescence line profile plot showing PTK2 ubiquitinated outside the PTK2 condensate. F. Western Blot and bar graphs show PTK2 levels reduction on KD-Bclaf1 cells treated with dox, while PTK2 mRNA expression levels increased. G. Western blot of soluble extracts from control and KD-Bclaf1 H9c2 cells treated with dox. Bar graphs show a reduction of PTK2-Full length (PTK2-FL) band and an increase in PTK2 cleaved bands (55 KDa and 34 kDa). H. Western blot and bar graph analysis of insoluble extract from control and KD-Bclaf1 H9c2 cells treated with dox. The analysis showed no difference between CT and KD-Bclaf1 cells on the amount of PTK2-FL and PTK2 fragments present on the insoluble fraction. I. Western blot and bar graphs show increased p53 and PUMA levels on KD-Bclaf1 H9c2 cardiomyocytes treated with dox. On the right, the bar graph shows a reduced number of cells on the KD-Bclaf1 cells in relation to control cells, after dox treatment.

Article Snippet: Other combinations were performed with antibodies against phosphorylated PTK2 (Thermo Fisher, 44-625G – 1:200) and Hsp70 (Thermo Fisher, MA3-006 – 1:50).

Techniques: Western Blot, Knockdown, Control, Ubiquitin Proteomics, Fluorescence, Expressing