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: Frontiers in Microbiology

Article Title: Candida albicans : The Ability to Invade Epithelial Cells and Survive under Oxidative Stress Is Unlinked to Hyphal Length

doi: 10.3389/fmicb.2017.01235

Figure Lengend Snippet: C. albicans SC5314 strain induced host cell biphasic ERK 1/2 activation and cortactin phosphorylation by SFKs. HeLa cells were incubated with blastospores from the SC5314 strain for the indicated time points. After incubation, HeLa cells were harvested, lysed, and their protein lysates separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blot using the indicated antibodies. The SC5314 strain induced (A) a biphasic activation of host ERK 1/2 (the other isolates did not, not shown) and also (B) the phosphorylation of cortactin (pCort [Y466]) by SFKs. These are representative observations from at least three independent experiments. SC5314, 997,5 g, oral L3837 and blood L3881 isolates induced host cell signaling activation. HeLa cells were incubated with blastospores from four C. albicans isolates for the indicated time points. After incubation, HeLa cells were harvested, lysed, and SFK and cortactin activation were analyzed by Western blot using the indicated antibodies. Cortactin, SFKs and actin have different molecular weights (80–85, 60, and 45 kDa, respectively), thus the nitrocellulose membrane was cut into three horizontal strips, blocked and each strip was incubated with anti-pY466 cortactin, anti-pY416 SFK or anti-actin. (C) pSFK (Y416) and (D) pCort (Y466), cortactin phosphorylated by Src at the Y 466 residue. Actin was used for protein loading normalization. Actin labeling is duplicated in (C,D) . pSFK/Act = densitometric rate of pSFK over actin. pCort/Act = densitometric rate of pCort over actin. These are representative observations from at least three independent experiments.

Article Snippet: Rabbit anti-pT202/Y204 ERK1/2 (#9101S) and rabbit anti-pY416 SFK (#2101S) were obtained from Cell Signaling, Beverly, MA, USA.

Techniques: Activation Assay, Phospho-proteomics, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Membrane, Stripping Membranes, Residue, Labeling

Journal: Neuroscience

Article Title: Linkage of non-receptor tyrosine kinase Fyn to mGlu5 receptors in striatal neurons in a depression model

doi: 10.1016/j.neuroscience.2020.02.048

Figure Lengend Snippet: (A) Effects of social isolation on pY416, Fyn, and Src levels in the CPu. (B) Effects of social isolation on pY416, Fyn, and Src levels in the NAc. Note that social isolation (SI) had no significant effect on phosphorylation and expression of Fyn and Src in the CPu (A) and NAc (B) as compared to control (Con) rats. Representative immunoblots are shown left to the quantified data. Data were statistically analyzed using Student’s t-test (n = 6 per group) with ‘n’ equal to the number of animals.

Article Snippet: A rabbit antibody against pY416 (RRID:AB_10013641; Cell Signaling) reacts with the SFK members when phosphorylated at a conserved activation residue, a pan Y416 site.

Techniques: Isolation, Expressing, Western Blot

Journal: The Journal of Cell Biology

Article Title: Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex

doi: 10.1083/jcb.201408103

Figure Lengend Snippet: VEcad TMD in flow signaling. (A) Requirement for VEcad. HUVECS were infected with scrambled or anti-VEcad shRNA-containing lentiviruses then subjected to 12 dynes/cm 2 laminar shear for 1 min. Activation of SFKs (SFK pY416 , ∼55 kD) and VEGFR2 (VEGFR2 pY1175 , ∼250 and 220 kD) were assayed by immunoblotting, with actin as a loading control. (B) VEcad TMD requirement for VEGFR activation. VEcad −/− cells reconstituted with VEcad WT , Ncad VE-TMD , Ncad WT , and VEcad N-TMD were subjected to laminar shear stress for 1 min, then VEGFR2 activation was assayed as in A. (C) VEcad requirement for PI3K signaling. Cells were subjected to laminar shear stress, then p85 immunoprecipitated and immunoblotted with anti-p85 pY458 antibody. Values beneath each panel indicate phosphorylation relative to cells without flow, quantified by densitometry with total p85 serving as a loading control. For all panels, values are means ± SEM, n = 3. IB, immunoblotting.

Article Snippet: Other phospho-antibodies used for immunoblotting include rabbit anti-SFK pY416 (#6943; Cell Signaling Technology), rabbit anti-p85 pY458 (#4228; Cell Signaling Technology), rabbit anti-Akt pS473 (700392; Invitrogen), and rabbit ant-p65 pS536 (#3033; Cell Signaling Technology).

Techniques: Infection, shRNA, Shear, Activation Assay, Western Blot, Control, Immunoprecipitation, Phospho-proteomics

Journal: Journal of Clinical Immunology

Article Title: A Novel Biallelic LCK Variant Resulting in Profound T-Cell Immune Deficiency and Review of the Literature

doi: 10.1007/s10875-023-01602-8

Figure Lengend Snippet: Reduced protein expression and TCR signaling due to LCK C465R. A LCK and GAPDH immunoblots of whole cell lysates of patient or healthy control (HC) T-cell blasts unstimulated or stimulated with anti-CD3 (2 min), anti-CD3/CD28 (2 min), PMA/ionomycin (P/I, 5 min) or treated with the selective LCK-inhibitor A770041 (A77, 10 min). Orange arrowheads indicate LCK bands. B pZAP70, pSFK (pY416), pERK1/2 or GAPDH immunoblots, conditions as in A . Red arrowheads indicate pLCK bands, black arrowheads pFYN-T. Numbers below pERK1/2 blot indicate fold change pERK1/2 band intensity (HC unstimulated =1), intensity normalized to GAPDH loading (numbers below GAPDH blot; GAPDH signal intensity normalized to HC unstimulated) C LCK, HA-tag and GAPDH immunoblots of whole cell lysates of Jurkat E6.1, J.CaM1.6, and stable transduced cells line expressing either LCK WT or LCK C465R with or without on C-terminal HA-tag or transduced with pCDH containing only the expression cassette for the extracellular domain of the low-affinity nerve growth factor receptor (LNGFR) (J.CaM EV). D Overlay of Ca 2+ -flux measurement in the indicated cell lines. Experiment representative of 3 independent experiments. E pZAP70, pSFK (pY416), pERK1/2 immunoblots in J.CaM1.6 cells transduced with pLJM1 containing LCK WT or LCK C465R and stimulated with either anti-CD3 (2, 5 or 15 min), anti-CD3/CD28 (2 or 5 min), PMA/ionomycin (5 min) or left untreated

Article Snippet: After a blocking step with 3% BSA TBS-T, immunoblotting was performed with the following antibodies: mouse anti-human FYN (clone E-3), mouse anti-human GAPDH (clone 6C5), mouse anti-human LCK (clone 3A5), mouse-IgGκ BP-HRP, rabbit-IgGκ BP-HRP, anti-mouse-IgG-HRP (all Santa Cruz Biotechnology) or rabbit anti-human pZAP70 pY319 (clone 65E4), rabbit anti-human polyclonal pSRC-family kinase (pSFK) pY416, rabbit anti-human pERK1/2 pT202/pY204 (clone 197G2), and rabbit anti-HA-tag (clone C29F4) (all Cell Signaling Technologies).

Techniques: Expressing, Western Blot, Control, Transduction

Journal:

Article Title: Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading

doi: 10.1128/MCB.22.8.2427-2440.2002

Figure Lengend Snippet: Src kinase activity is needed for integrin-dependent FAK phosphorylation. SYF cells were infected using a retroviral system to stably express wtSrc or KD-Src mutants (K295R or D386A) or were infected with virus containing empty vector. Cells were harvested with trypsin, washed, and lysed with Triton buffer in suspension (0 min) or after plating for the indicated times (10, 20, or 30 min) on FN-coated dishes. FAK tyrosine phosphorylation was examined by immunoprecipitating with anti-FAK C20, followed by Western blotting with either anti-FAK C20 to demonstrate equal amounts of FAK in each immunoprecipitate (A), anti-phosphotyrosine 4G10 (B), or the site-specific phosphoantibodies anti-FAK-pY576 (C) or anti-FAK-pY397 (D). Src expression levels were determined by Western blotting cell lysates with anti-Src LA074 (E).

Article Snippet: The site-specific rabbit polyclonal phosphoantibodies anti-Src pY418 (here called anti-Src pY416 to denote chicken c-Src numbering), anti-FAK pY397, and anti-FAK pY576 were purchased from Biosource International, Inc. Generation of Src point mutations. pLXSH retroviral vectors containing wt chicken c-Src, the KD K295R mutant, or the activated Y527F mutant were described previously ( 4 , 38 ). pGEX vectors encoding glutathione transferase (GST) fusion proteins with the SrcSH2 ( 14 ) or SrcSH3 ( 4 ) domain were also described previously.

Techniques: Activity Assay, Infection, Stable Transfection, Plasmid Preparation, Western Blot, Expressing

Journal:

Article Title: Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading

doi: 10.1128/MCB.22.8.2427-2440.2002

Figure Lengend Snippet: Regulated Src kinase activity is not required for FAK phosphorylation upon integrin activation. SYF cells expressing the indicated Src molecules (or reconstituted with empty vector) were harvested with trypsin, washed, and lysed in suspension (0 min) or after plating on FN-coated dishes for 10 or 30 min, as indicated. (A and B) Src was immunoprecipitated from RIPA buffer lysates with anti-Src LA074, followed by Western blotting with anti-Src-pY416 (A) or anti-Src SRC2 (B). (C and D) FAK was immunoprecipitated from Triton buffer lysates with anti-FAK C20 followed by Western blotting with anti-phosphotyrosine 4G10 (C) or anti-FAK C20 (D).

Article Snippet: The site-specific rabbit polyclonal phosphoantibodies anti-Src pY418 (here called anti-Src pY416 to denote chicken c-Src numbering), anti-FAK pY397, and anti-FAK pY576 were purchased from Biosource International, Inc. Generation of Src point mutations. pLXSH retroviral vectors containing wt chicken c-Src, the KD K295R mutant, or the activated Y527F mutant were described previously ( 4 , 38 ). pGEX vectors encoding glutathione transferase (GST) fusion proteins with the SrcSH2 ( 14 ) or SrcSH3 ( 4 ) domain were also described previously.

Techniques: Activity Assay, Activation Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal:

Article Title: Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading

doi: 10.1128/MCB.22.8.2427-2440.2002

Figure Lengend Snippet: Src is not localized to focal adhesions, while localization of other focal adhesion proteins is independent of SFK expression. SYF cells reconstituted with vector (B and D), wtSrc (A, C, E, and G), or Y416F Src mutant (F and H) were harvested with trypsin, washed, and plated on FN-coated coverslips in 0.5% FBS for 1 h. Cells were washed, fixed, permeabilized and stained with MAb anti-FAK (A and B) or anti-Src LA074 (C and D), followed by FITC-conjugated anti-mouse secondary antibody. Some cells were costained with MAb anti-paxillin (E and F) and anti-Src pY416 (G and H), followed by Texas Red and FITC-conjugated secondary antibodies to detect paxillin and active Src, respectively. The faint nuclear staining seen with anti-Src pY416 (G and H) is due to nonspecific bleed-through from DAPI staining (not shown). Scale bar, ∼50 μm.

Article Snippet: The site-specific rabbit polyclonal phosphoantibodies anti-Src pY418 (here called anti-Src pY416 to denote chicken c-Src numbering), anti-FAK pY397, and anti-FAK pY576 were purchased from Biosource International, Inc. Generation of Src point mutations. pLXSH retroviral vectors containing wt chicken c-Src, the KD K295R mutant, or the activated Y527F mutant were described previously ( 4 , 38 ). pGEX vectors encoding glutathione transferase (GST) fusion proteins with the SrcSH2 ( 14 ) or SrcSH3 ( 4 ) domain were also described previously.

Techniques: Expressing, Plasmid Preparation, Mutagenesis, Staining

Journal:

Article Title: Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading

doi: 10.1128/MCB.22.8.2427-2440.2002

Figure Lengend Snippet: Activity of Src SH2 and SH3 domain mutants. SYF cells stably expressing wtSrc, an SH2 domain mutant (T215W) or an SH3 domain mutant (D99N), or an activated mutant (Y527F) were established. (A) Phase-contrast images of near-confluent cell cultures at ×100 magnification (scale bar, ∼200 μm). (B) Lysates using Triton buffer were generated from similar cultures, and total proteins were Western blotted with anti-phosphotyrosine MAb 4G10. The positions of molecular mass markers (in kDa) are shown on the right. An asterisk indicates the position of Src. To detect the activation level of each form of Src, anti-Src immunoprecipitates (using MAb LA074) from growing cells were Western blotted with anti-Src-pY416 (C) or anti-Src LA074 (D).

Article Snippet: The site-specific rabbit polyclonal phosphoantibodies anti-Src pY418 (here called anti-Src pY416 to denote chicken c-Src numbering), anti-FAK pY397, and anti-FAK pY576 were purchased from Biosource International, Inc. Generation of Src point mutations. pLXSH retroviral vectors containing wt chicken c-Src, the KD K295R mutant, or the activated Y527F mutant were described previously ( 4 , 38 ). pGEX vectors encoding glutathione transferase (GST) fusion proteins with the SrcSH2 ( 14 ) or SrcSH3 ( 4 ) domain were also described previously.

Techniques: Activity Assay, Stable Transfection, Expressing, Mutagenesis, Generated, Western Blot, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: The Pattern of Enhancement of Src Kinase Activity on Platelet-derived Growth Factor Stimulation of Glioblastoma Cells Is Affected by the Integrin Engaged

doi: 10.1074/jbc.m304685200

Figure Lengend Snippet: FIG. 9. PDGF stimulation results in Fyn activation in U-87MG cells adherent to laminin. A and B, serum-starved U-87MG cells were harvested with buffered EDTA, resuspended in serum-free media and plated onto laminin (LM) or collagen (COL)-coated plates (5 h), and then stimulated with 83 pM PDGF or vehicle (10 min, 37 °C, 5% CO2), lysed, 300 g of lysate immunoprecipitated with the antibody indicated, the immunoprecipitates subjected to SDS-PAGE, transferred to Immo- bilon and blotted with anti-Src[pY416] IgG (A), stripped, and reprobed with anti-Lyn IgG or anti-Fyn IgG (B). C and D, U-87MG cells were harvested and resuspended as in Fig. 1, incubated with 20 g/ml neutralizing anti-integrin antibody (20 min, 22 °C), and then plated onto a 96-well plate previously coated with collagen or laminin and allowed to attach for 60 min (37 °C, 5% CO2). Wells were washed two times with PBS, the cells harvested with trypsin and counted in a scintillation counter. Attachment to ovalbumin was subtracted. Condi- tions were assayed in replicas of five and the data analyzed and pre- sented as the mean S.E. The experiment was repeated two times.

Article Snippet: The following rabbit antibodies were purchased: anti-phosphospecific Src [pY416] IgG (Santa Cruz Biotechnology, Inc.) (detects only activated Src), anti-phosphospecific Src[pY418] IgG (BioSource International) (detects only activated Src), anti-FAK (focal adhesion kinase) IgG (Upstate Biotechnology Inc.), anti-phosphospecific FAK(pY397) IgG (BioSource International), anti-PDGFr IgG (Santa Cruz Biotechnology, Inc.), anti-PDGFr IgG (Santa Cruz Biotechnology, Inc.), and anti-PDGFr IgG that recognizes both receptor forms (Upstate Biotechnology Inc.).

Techniques: Activation Assay, Immunoprecipitation, SDS Page, Incubation