Journal: Life Science Alliance

Article Title: Tyrosine phosphorylation of lamin A by Src promotes disassembly of nuclear lamina in interphase

doi: 10.26508/lsa.202101120

Figure Lengend Snippet: (A) MCF7 and MDA-MB-231 cells were treated with (+) or without (−) 50 nM dasatinib for 1 h and then lysed. Equal amounts of the whole cell lysates (WCLs) were incubated with anti-lamin A/C or pre-immune serum (IgG) as the control. The immunonocomplexes were analyzed by immunoblotting (IB) with anti-PY or anti-lamin A/C antibodies. An equal amount of WCLs was analyzed by immunoblotting with anti-Src, anti-Src pY416, or anti-actin. The tyrosine phosphorylation of lamin A was quantified and expressed as -fold relative to the level of MCF7 without dasatinib. (B) MCF7 and MDA-MB-231 cells were treated with (+) or without (−) 50 nM dasatinib for 1 h. The cells were fixed and stained for lamin A, lamin B1, and DNA. Representative images are shown. Scale bars, 10 μm. The nuclear circularity (4π × area/perimeter 2 ) was determined. The P -values were calculated from at least 150 cells pooled from three independent experiments. The percentage of the cells with nuclear lobulation was measured (n ≥ 400). The values (mean ± SD) are from three experiments. *** P < 0.001. (C) MDA-MB-231 cells were infected with lentivirues capable of expressing FLAG-lamin A or the mutants (Y45F and Y45D) and selected in the medium with neomycin (neo). An equal amount of WCLs was analyzed by immunoblotting (IB) with antibodies as indicated. (D) The cells as described in panel C were fixed and stained for FLAG-lamin A, lamin B1, and DNA. Representative images are shown. Scale bars, 10 μm. The percentage of the cells with nuclear lobulation was measured (n ≥ 900). Values (means ± SD) are from three experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are available for this figure.

Article Snippet: The rabbit polyclonal anti-Src pY416 (MAB2685) antibody was purchased from R&D Systems.

Techniques: Incubation, Control, Western Blot, Phospho-proteomics, Staining, Infection, Expressing

Journal: Cancers

Article Title: Neurofibromin Deficiency and Extracellular Matrix Cooperate to Increase Transforming Potential through FAK-Dependent Signaling

doi: 10.3390/cancers13102329

Figure Lengend Snippet: Neurofibromin loss sensitizes MEF cells to growth factor and matrix stimulation: ( a ) MEF cells isolated and immortalized from wild-type and knockout mice for Nf1 gene were analyzed by Western blotting with anti-neurofibromin (Nfn) antibodies; ( b ) focal adhesion kinase FAK binds to Nfn. FAK was immunoprecipitated (IP) from cell lysates of wild-type and knockout MEFs ( Nf1 +/+ , Nf1 −/− ); following SDS-PAGE electrophoresis, co-immunoprecipitated Nfn were assayed by Western blotting analysis (IB) with anti-FAK and anti-Nfn antibodies. Total protein loaded on the IP is also shown as INPUT (( b ), lower panel) and examined by anti-Nfn and anti-actin antibodies; ( c ) transient phosphorylation kinetics of MEFs stimulated with PDGF-BB (10 ng/mL), [ , ] for increasing time periods. Following SDS-PAGE, cell lysates were incubated with anti-phospho-Erk (1/2) (T202/Y204, T185/Y187) MAP kinase, phospho-FAK (Y397) and phospho-Src (Y416) antibodies and anti-GAPDH; ( d , e ) colony formation assay of MEFs fully embedded by low growth factor containing Matrigel in absence (upper panel) or presence (lower panel) of insoluble matrix proteins Collagen I/Fibronectin. Cells were seeded in the Matrigel and 24 h later treated or not with PDGF-BB ligand for 8 days (representative microscope pictures (10×)). Where indicated, MEF cells were additionally treated with FAK and MEK inhibitors named Y10 (0.62 μM) and Selumetinib (6.62 μM), respectively; the area of colonies was calculated in five fields for each well, and mean value and SD calculated and plotted in the right histograms. n = 8; * p < 0.05 **; p < 0.001 by Student’s t -test.

Article Snippet: Actin (C-11) (sc-1615) from Santa Cruz Biotechnology (Heidelberg, Germany), mouse anti-Akt (#2920, 40D4), rabbit anti-phospho-Akt (S473, #9271), rabbit anti-Src (Y416) anti-GAPDH (#2118, Cell Signaling), rabbit anti-phospho-p44/42 ERK (#9101), anti-phospho-FAK (Y925 #3284), anti-FAK (#3285) were purchased from (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-FAK (Y397) (44-624G, Invitrogen, Carlsbad, CA, USA).

Techniques: Isolation, Knock-Out, Western Blot, Immunoprecipitation, SDS Page, Electrophoresis, Phospho-proteomics, Incubation, Colony Assay, Microscopy

Journal: Cancer cell

Article Title: KRAS Suppression-Induced Degradation of MYC is Antagonized by a MEK5-ERK5 Compensatory Mechanism

doi: 10.1016/j.ccell.2018.10.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cat# 4290 Rabbit anti-phospho-Src Family Y416 Cell Signaling Tech.

Techniques: Recombinant, In Vivo, Viability Assay, Software