p38 mouse mab  (Proteintech)

Journal: bioRxiv

Article Title: Adhesion-derived condensates control component availability to regulate adhesion dynamics

doi: 10.1101/2025.05.08.652869

Figure Lengend Snippet: A) Representative timelapse images of either non-treated U2OS cells expressing GFP-TNS1 (CTRL) or cells treated with sodium arsenite (ARS). Images were acquired for 60 min at 1 min intervals. Images at t = 0 min and t = 60 min are shown, along with a kymograph of the whole timelapse along the white line. Scale bars 20 μm. B) Quantification of the ratio between the relative cytoplasmic and droplet TNS1 integrated densities at the indicated timepoints from n = 78-115 cells per condition. Statistical analysis was performed using Friedman test with Dunn’s multiple comparisons test comparing the individual timepoints to t = 0 min. p < 0.001 (***); ns. – not significant. C) Representative immunoblots from lysates of either CTRL or ARS-treated cells. GFP-TNS1 was immunoprecipitated to probe with anti-pSer/pThr antibody. D) Quantification of relative TNS1 Ser/Thr phosphorylation and relative activity of p38, ERK and AKT kinases quantified as ratio between phosphorylated and total protein. Quantification was performed from three biological replicates. Statistical analysis was performed using unpaired t-test. p < 0.01 (**); p < 0.001 (***). E) Representative timelapse images of U2OS cells expressing GFP-TNS1 pre-treated with 5 μM Doramapimod (p38), 5 μM Trametinib (MEK1/2) and 5 μM MK2206 (Akt) for 60 min before ARS treatment. Images were acquired for 60 min at 1 min intervals. Images at t = 0 min and t = 60 min are shown, along with a kymograph of the whole timelapse along the white line. Scale bars 20 μm. F) Quantification of the ratio between the relative cytoplasmic and droplet TNS1 integrated densities at t = 60 min from n = 78-115 cells per condition. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparisons test. p < 0.001 (***); ns. – not significant. G) Volcano plot of individual TNS1 phosphorylation sites as identified by mass spectrometry of GFP-TNS1 purified from CTRL or ARS-treated cells. Statistically enriched (> 2-fold change at p < 0.05) Ser/Thr phosphosites are indicated in yellow and Tyr phosphosites in blue. H) Schematic representation of localisation of the identified phosphosites within TNS1. I) Representation of the cumulative phosphosite number over 30 amino acid (AA) window within TNS1 purified from CTRL or ARS-treated cells. J) Representative confocal images of U2OS TNS1 KO cell lines with inducible expression of indicated GFP-TNS1 variants. Scale bars 20 μm. K) Quantification of TNS1 droplet count per cell as quantified from n = 339-358 cells per condition. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparisons test. p < 0.001 (***). L) Quantification of average TNS1 droplet size per cell as quantified from n = 340-358 cells per condition. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparisons test. p < 0.001 (***).

Article Snippet: Primary antibodies used were as follows: anti-GFP (1:5000, Abcam, ab290); GAPDH (1:4000, HyTest, 5G4 MAb 6C5); anti-TNS1 (1:1000, Sigma, SAB4200283); anti-ZYX (1:1000, Abcam, ab109316); anti-PXN (1:1000, GeneTex, GTX125891); anti-TLN1 (1:1000, Novus Biologicals, NBP2-50320); anti-VCL (1:1000, Sigma, V9131); anti-Myc-Tag (1:1000, CST, 2276S); anti-ITGB1 (1:1000, BD Transduction Laboratories, 610468); anti-ITGA5 (1:1000, Invitrogen, PA5-82027); anti-pSer/Thr (1:1000, BD Transduction Laboratories, 612549); anti-p38 (1:1000, CST, 9212S); anti-p-p38 (1:1000, CST, 9216S); anti-AKT (1:1000, CST, 2920S); anti-p-AKT (1:1000, CST, 9275S); anti-ERK (1:1000, CST, 4696S); anti-p-ERK (1:1000, CST, 4370S).

Techniques: Expressing, Western Blot, Immunoprecipitation, Phospho-proteomics, Activity Assay, Mass Spectrometry, Purification

Journal: bioRxiv

Article Title: Adhesion-derived condensates control component availability to regulate adhesion dynamics

doi: 10.1101/2025.05.08.652869

Figure Lengend Snippet: A) Quantification of normalized relative average TNS1 droplet size from n = 81-119 cells analysed in . Statistical analysis was performed using Friedman test with Dunn’s multiple comparisons test comparing the individual timepoints to t = 0 min. p < 0.001 (***); ns. – not significant. B) Quantification of normalized relative TNS1 droplet area from n = 80-117 cells analysed in . Statistical analysis was performed using Friedman test with Dunn’s multiple comparisons test comparing the individual timepoints to t = 0 min. p < 0.001 (***); ns. – not significant. C) Quantification of the ratio between the relative cytoplasmic and droplet integrated densities of GFP-Pop, GFP-TNS1 and GFP-TNS2 at t = 60 min from n = 104-115 cells per condition. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparisons test. p < 0.05 (*); p < 0.001 (***). D) Representative timelapse images of ARS-treated U2OS cells expressing either GFP-Pop, GFP-TNS1 or GFP-TNS2. Images were acquired for 60 min at 1 min intervals. Images at t = 0 min and t = 60 min are shown. Scale bars 20 μm. E) Quantification of the ratio between the relative cytoplasmic and droplet GFP-TNS2 integrated densities at the indicated timepoints from n = 112 cells. Statistical analysis was performed using Friedman test with Dunn’s multiple comparisons test comparing the individual timepoints to t = 0 min. p < 0.05 (*); p < 0.01 (**); ns. – not significant. F) Quantification of the ratio between the relative cytoplasmic and droplet GFP-Pop integrated densities at the indicated timepoints from n = 104 cells. Statistical analysis was performed using Friedman test with Dunn’s multiple comparisons test comparing the individual timepoints to t = 0 min. p < 0.01 (**); ns. – not significant. G) Plot of normalized TNS1 quantities identified by mass spectrometry. H) Cumulative net charge distribution over 30 AA window in TNS1 from CTRL (left) or ARS-treated (right) cells. I) Heatmap of phosphorylation probability score for individual phosphorylation sites by p38α, ERK1 and AKT1 kinases as predicted by Kinase Library tool . Top panel represents Log 2 fold-change of individual TNS1 phosphosites in CTRL and ARS-treated conditions. Bottom panel (SD/SG substitution) indicates phosphorylation sites selected for molecular dynamics simulations and experimental validation. J) Representative immunoblots from lysates of U2OS cells with inducible expression of the indicated GFP-TNS1 variants. K) Quantification of integrated density of cells analysed in and . Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparisons test. p < 0.05 (*); ns. – not significant. L) Quantification of TNS1 droplet area per cell as quantified from n = 343-358 cells per condition. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparisons test. p < 0.01 (**); p < 0.001 (***).

Article Snippet: Primary antibodies used were as follows: anti-GFP (1:5000, Abcam, ab290); GAPDH (1:4000, HyTest, 5G4 MAb 6C5); anti-TNS1 (1:1000, Sigma, SAB4200283); anti-ZYX (1:1000, Abcam, ab109316); anti-PXN (1:1000, GeneTex, GTX125891); anti-TLN1 (1:1000, Novus Biologicals, NBP2-50320); anti-VCL (1:1000, Sigma, V9131); anti-Myc-Tag (1:1000, CST, 2276S); anti-ITGB1 (1:1000, BD Transduction Laboratories, 610468); anti-ITGA5 (1:1000, Invitrogen, PA5-82027); anti-pSer/Thr (1:1000, BD Transduction Laboratories, 612549); anti-p38 (1:1000, CST, 9212S); anti-p-p38 (1:1000, CST, 9216S); anti-AKT (1:1000, CST, 2920S); anti-p-AKT (1:1000, CST, 9275S); anti-ERK (1:1000, CST, 4696S); anti-p-ERK (1:1000, CST, 4370S).

Techniques: Expressing, Mass Spectrometry, Phospho-proteomics, Biomarker Discovery, Western Blot