Phospho-Lamin A/C (Ser301) Antibody - #AF7177

Fig. 4: Loss of AR compromises the association of lamin A/C with the phosphatase PPP1 and results in increased lamin A/C phosphorylation at Ser 301. A Cytoscape network of proteins with reduced association with lamin A/C in AR-silenced HDFs. Proteins were identified by LC-MS/MS analysis from three different HDF strains with/without AR silencing. The list of interactors is shown in Supplementary Data S1. Names of proteins with reduced association (log fold change >1.5) in at least two strains are displayed. The inset shows PPP1 subunits associated with lamin A/C in an AR-dependent manner. B Co-immunoprecipitation assays with HDFs plus/minus AR silencing with anti-lamin A/C antibodies or nonimmune IgGs followed by immunoblotting with anti-AR, -lamin A/C, and -PPP1CA/B antibodies, with parallel analysis of the inputs. C PLA with anti-lamin A/C and PPP1CA/B antibodies of HDFs plus/minus AR silencing. Shown are representative images and quantification of the number of puncta per cell, with the mean value as red bar. Non-parametric one-way ANOVA, n = 53 cells per condition. Scale: 10 µm. D Immunoblot analysis of HDFs treated with ARCC4 (1 µM) or AZD3514 (10 µM) for 72 h, with phospho-Ser301 lamin A/C, total lamin A/C, and β-Actin antibodies. E Immunofluorescence analysis of three HDF strains treated with ARCC4 (1 µM) or EtOH for 72 h, antibodies against phospho-Ser301-lamin A/C (yellow), and DAPI for nuclear staining. Representative images (left) and fluorescence signal intensity quantification (right), Two-tailed unpaired t-test, n = 3 strains (50 cells per condition), mean ± SE. Scale: 10 µm. F IF analysis of three HDF strains, infected with AR-silencing lentiviruses versus control, with antibodies against phospho-Ser301(pSer301)-lamin A/C (yellow) and counterstained with DAPI. Representative images (left) and fluorescence intensity quantification (right), nonparametric one-way ANOVA, n = 3 strains (50 cells per condition), mean ± SE. Scale: 10 µm. Fluorescence intensities per individual counted cells for each strain are shown in Supplementary Fig. 4D. G 3D Surface Reconstruction: Whole nuclei of HDFs plus/minus AR silencing stained with anti-phospho-S301-lamin A/C (red) and total lamin A/C (yellow). Z-stacks of confocal images used for 3D volumetric reconstruction with Imaris Surface tool. 2D and 3D images of Lamin A/C and pSer301 signals; top and lateral views. Scale: 5 µm. Source data for individual graphs are in the Source Data file.

Figure 5. Lamin A/C phosphorylation at Ser 301 is a signature of CAFs. A) IF analysis of CAFs and matched normal fibroblasts (HDFs) from the same patient with images of pSer301 lamin A/C (pSer301; red) and total lamin A/C (yellow). Shown are representative images and quantification of phospho-ser301-lamin A/C fluorescence signal intensity assessed by Image J and expressed in arbitrary units (AU). n(cells)>50 per condition together with mean and statistical significance, ****p50 per condition together with mean and statistical significance, ****p20 per together with mean and statistical significance, ****p50 per condition together with mean and statistical significance, ****p