rabbit polyclonal anti-src-1 sc-8995  (Santa Cruz Biotechnology)

Journal: Reproductive biomedicine online

Article Title: METTL3 and METTL14-mediated N 6 -methyladenosine modification promotes cell proliferation and invasion in a model of endometriosis.

doi: 10.1016/j.rbmo.2022.10.010

Figure Lengend Snippet: FIGURE 3 Validation of the N6-methyladenosine (m6A) methylation level and mRNA level of the genes enriched in estrogen signalling. (A) The m6A methylation level of MMP2, NCOA1, TLR4, PPP2R2D and YWHAZ in the ectopic endometrium (black bar) and eutopic endometrium (EU) (hatched gray bar) was validated using MeRIP quantitative polymerase chain reaction; (B) the expression of MMP2, NCOA1, TLR4, PPP2R2D and YWHAZ in the ectopic endometrium (black bar) and eutopic endometrium (hatched gray bar) using quantitative reverse transcription polymerase chain reaction; and (C) immunohistochemistry was carried out to assess the protein levels of MMP2, NCOA1, PPP2R2D and YWHAZ in the ectopic endometrium and eutopic endometrium; magnification, × 200. Two-tailed Student's t-tests were used for comparisons between ectopic endometrium and eutopic endometrium. All samples were assayed in triplicate.

Article Snippet: The polyclonal rabbit immunoglobulin G (IgG) antibody against human NCOA1 (51114-1-AP) (ProteinTech, Rosemont, IL, USA), polyclonal rabbit IgG antibody against human MMP2 (10373-2-AP) (ProteinTech, Rosemont, IL, USA), polyclonal rabbit antibody against human PPP2R2D (bs-19968P) (BiosSS Woburn, MA, USA), polyclonal rabbit IgG antibody against human YWHAZ (14881-1-AP) (Proteintech, Sankt LeonRot, Germany), were diluted at 1:1000.

Techniques: Biomarker Discovery, Methylation, Real-time Polymerase Chain Reaction, Expressing, Reverse Transcription, Polymerase Chain Reaction, Immunohistochemistry, Two Tailed Test

Journal: Reproductive biomedicine online

Article Title: METTL3 and METTL14-mediated N 6 -methyladenosine modification promotes cell proliferation and invasion in a model of endometriosis.

doi: 10.1016/j.rbmo.2022.10.010

Figure Lengend Snippet: FIGURE 5 Knockdown of METTL3, METTL14, or both, contributes to the cellular phenotype of endometriosis. (A–B) Quantitative polymerase chain reaction and Western blotting analysis of the expression of NCOA1, MMP2 For photomicrographs, please provide scale bars. PPP2R2D and YWHAZ after the knockdown of METTL3 and(or) METTL14. Cell viability assay of the cells with METTL3 and METTL14 knockdown; (C) the CCK-8 assay of the cell viability after the knockdown of METTL3 and METTL14; (D) the scratch assay of the cell migration after the knockdown of METTL3 and METTL14; (E) transwell assay of the effect of the METTL3 and METTL14 knockdown on the invasion and migration of endometrial stromal cells. NC: negative control; si-METTL14 was transfected with siMETTL14; si-METTL3 was transfected with siMETTL3; si-METTL3+si-METTL14 were transfected with si-METTL3 and si-METTL14. All samples were assayed in triplicate.

Article Snippet: The polyclonal rabbit immunoglobulin G (IgG) antibody against human NCOA1 (51114-1-AP) (ProteinTech, Rosemont, IL, USA), polyclonal rabbit IgG antibody against human MMP2 (10373-2-AP) (ProteinTech, Rosemont, IL, USA), polyclonal rabbit antibody against human PPP2R2D (bs-19968P) (BiosSS Woburn, MA, USA), polyclonal rabbit IgG antibody against human YWHAZ (14881-1-AP) (Proteintech, Sankt LeonRot, Germany), were diluted at 1:1000.

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Viability Assay, CCK-8 Assay, Wound Healing Assay, Migration, Transwell Assay, Negative Control, Transfection

Journal: Apoptosis

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

doi: 10.1007/s10495-019-01576-2

Figure Lengend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p < 0.05 vs. the untreated samples)

Article Snippet: To determine the total and phosphorylated Src1, the membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) in TBST and a rabbit monoclonal anti-human phospho-Tyr416 Src-family antibody (D49G4), diluted 1:4000 (v/v) in TBST (both from Cell Signalling Technologies/New England Biolabs, Hitchin, UK).

Techniques: Phospho-proteomics, Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Journal: Apoptosis

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

doi: 10.1007/s10495-019-01576-2

Figure Lengend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p < 0.05 vs. the untreated samples)

Article Snippet: To determine the total and phosphorylated Src1, the membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) in TBST and a rabbit monoclonal anti-human phospho-Tyr416 Src-family antibody (D49G4), diluted 1:4000 (v/v) in TBST (both from Cell Signalling Technologies/New England Biolabs, Hitchin, UK).

Techniques: Phospho-proteomics, Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

Journal: Apoptosis

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

doi: 10.1007/s10495-019-01576-2

Figure Lengend Snippet: Analysis of Src phosphorylation, kinase activity and induction of cell apoptosis. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of pCMV6-Ac-tGFP, pCMV6-Ac-TF Ala253 -tGFP, pCMV6-Ac-TF-tGFP plasmids, along with an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) for 90 min. a The cells were then lysed and phosphorylated and total Src1 examined by western blot, as described before. b The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 4; * = p < 0.05 vs. the untreated samples). c HCAEC (2 × 10 5 /well) were transfected as above and incubated for 48 h to permit protein expression. Cells were then adapted to low-serum medium MV containing 2% (v/v) FCS for 60 min and activated with PAR2-AP (20 μM) for a further 90 min. The cells were then lysed in PhosphoSafe Extraction Reagent and the Src activity measured using the ProFluor ® Src-family kinase assay. The fluorescence intensity of the released rhodamine was measured at Em 530 nm (Ex 485 nm), using the absorption of the included AMC at Em 460 nm (Ex 355 nm), as reference for the initial substrate concentration. (n = 5; * = p < 0.05 vs. the untreated samples). d HCAEC (2 × 10 5 /well) were transfected as above and incubated for 48 h to permit protein expression. Cells were then adapted to low-serum medium MV containing 2% (v/v) FCS for 60 min and activated with PAR2-AP (20 μM) for a further 90 min. The rate of cellular apoptosis was then quantified at 20 h, using the TiterTACS™ Colorimetric Apoptosis Detection Kit. (n = 4; * = p < 0.05 vs. the untreated samples)

Article Snippet: To determine the total and phosphorylated Src1, the membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) in TBST and a rabbit monoclonal anti-human phospho-Tyr416 Src-family antibody (D49G4), diluted 1:4000 (v/v) in TBST (both from Cell Signalling Technologies/New England Biolabs, Hitchin, UK).

Techniques: Phospho-proteomics, Activity Assay, Transfection, Incubation, Expressing, Recombinant, Western Blot, Extraction, Kinase Assay, Fluorescence, Concentration Assay

Journal: Apoptosis

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

doi: 10.1007/s10495-019-01576-2

Figure Lengend Snippet: Examination of the involvement of Src1 in TF-mediated induction of cell apoptosis. Sets of HCAEC (2 × 10 5 /well) were transfected with pCMV6-Ac-TF Ala253 -tGFP and incubated for 48 h to permit protein expression. a Cells were then adapted to low-serum medium MV containing 2% (v/v) FCS for 60 min together with the Src1 inhibitor pp60c Src inhibitor (TSTEPQpYQPGENL; 500 µM) and compared to a pseudo inhibitor (TSTEPQWQPGENL 500 nM). The cells were then activated with PAR2-AP (20 μM) for a further 90 min. A set of cells was also treated with IL-1β (10 ng/ml) as a positive control. The rate of cellular apoptosis was then quantified at 20 h, using the TiterTACS™ Colorimetric Apoptosis Detection Kit. (n = 4; * = p < 0.05 vs. sample devoid of inhibitor). b Sets of cells were transfected as above and then adapted to low-serum medium MV containing 2% (v/v) FCS for 60 min together with a range of Src1 inhibitor pp60c (0–500 nM). The cells were then activated with PAR2-AP (20 μM) for a further 90 min. The rate of cellular apoptosis was then quantified at 20 h, using the TiterTACS™ Colorimetric Apoptosis Detection Kit. (n = 4; * = p < 0.05 vs. the sample devoid of inhibitor). c Sets of HCAEC (2 × 10 5 /well) were co-transfected with pCMV6-Ac-TF Ala253 -tGFP plasmid together with wither a Src1 siRNA, control siRNA or without any siRNA, and incubated for 48 h. The rate of cellular apoptosis was then quantified at 20 h, using the TiterTACS™ Colorimetric Apoptosis Detection Kit. (n = 4; * = p < 0.05 vs. sample devoid of siRNA). d HCAEC (2 × 10 5 ) were transfected with pCMV6-Ac-TF Ala253 -tGFP plasmid and incubated for 48 h to permit the expression of the recombinant protein. The cells were then activated with PAR2-AP (20 µM) and lysed and the amounts of phosphorylated-p38 and total-p38 were examined by western blot and e The ratios of phosphorylated to total p38 were calculated using the ImageJ program. (n = 4; * = p < 0.05 vs. sample devoid of inhibitor)

Article Snippet: To determine the total and phosphorylated Src1, the membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) in TBST and a rabbit monoclonal anti-human phospho-Tyr416 Src-family antibody (D49G4), diluted 1:4000 (v/v) in TBST (both from Cell Signalling Technologies/New England Biolabs, Hitchin, UK).

Techniques: Transfection, Incubation, Expressing, Positive Control, Plasmid Preparation, Control, Recombinant, Western Blot

Journal: Apoptosis

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

doi: 10.1007/s10495-019-01576-2

Figure Lengend Snippet: Examination of the involvement of FAK in TF-mediated induction of cell apoptosis. Sets of HCAEC (2 × 10 5 /well) were transfected with pCMV6-Ac-tGFP, pCMV6-Ac-TF Ala253 -tGFP, pCMV6-Ac-TF-tGFP plasmids, along with an untransfected set of cells and incubated for 48 h to permit protein expression. Cells were then adapted to low-serum medium MV containing 2% (v/v) FCS for 60 min together with FAK inhibitor-14 (100 µM) and compared to vehicle control. The cells were then activated with PAR2-AP (20 μM) for a further 90 min. a The cells were then lysed and phosphorylated and total Src1 examined by western blot, as described before. b The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 4; * = p < 0.05 vs. the untreated samples). c Phosphorylation of FAK was also analysed by western blot using specific antibodies to Tyr397-phosphorylated and total FAK. d The ratios of phosphorylated to total FAK were calculated using the ImageJ program. (n = 4; * = p < 0.05 vs. the untreated samples). e Sets of cells were treated as above and then lysed in PhosphoSafe Extraction Reagent and the Src kinase activity measured using the ProFluor ® Src-family kinase assay. (n = 4). f In addition, the rate of cellular apoptosis was then quantified at 20 h, using the TiterTACS™ Colorimetric Apoptosis Detection Kit. (n = 4). g Sets of HCAEC were transfected and adapted to serum-free medium as above. One set of cells were then activated with PAR2-AP (20 µM) and the level of FAK phosphorylation by western blot. h The ratios of phosphorylated to total FAK were calculated using the ImageJ program. (n = 4)

Article Snippet: To determine the total and phosphorylated Src1, the membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) in TBST and a rabbit monoclonal anti-human phospho-Tyr416 Src-family antibody (D49G4), diluted 1:4000 (v/v) in TBST (both from Cell Signalling Technologies/New England Biolabs, Hitchin, UK).

Techniques: Transfection, Incubation, Expressing, Control, Western Blot, Phospho-proteomics, Extraction, Activity Assay, Kinase Assay

Journal: Apoptosis

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

doi: 10.1007/s10495-019-01576-2

Figure Lengend Snippet: Examination of the involvement of β1-integrin in TF-mediated induction of cell apoptosis. Sets of HCAEC (2 × 10 5 /well) were transfected with pCMV6-Ac-tGFP, pCMV6-Ac-TF Ala253 -tGFP, pCMV6-Ac-TF-tGFP plasmids, along with an untransfected set of cells and incubated for 48 h to permit protein expression. Cells were then adapted to low-serum medium MV containing 2% (v/v) FCS for 60 min together with a blocking anti-β1-integrin antibody (AIIB2; 20 µg/ml) and then were activated with PAR2-AP (20 μM) for a further 90 min. a The cells were then lysed and phosphorylated and total Src1 examined by western blot, as described before. b The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 4; * = p < 0.05 vs. untreated samples; # = p < 0.05 vs. respective control IgG). c Sets of cells were treated as above and then lysed in PhosphoSafe Extraction Reagent and the Src kinase activity measured using the ProFluor ® Src-family kinase assay. (n = 4; * = p < 0.05 vs. the untreated samples; # = p < 0.05 vs. respective control IgG). d Sets of cells were treated as above and the rate of cellular apoptosis was quantified at 20 h, using the TiterTACS™ Colorimetric Apoptosis Detection Kit. (n = 4; * = p < 0.05 vs. the untreated samples; # = p < 0.05 vs. respective control IgG)

Article Snippet: To determine the total and phosphorylated Src1, the membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) in TBST and a rabbit monoclonal anti-human phospho-Tyr416 Src-family antibody (D49G4), diluted 1:4000 (v/v) in TBST (both from Cell Signalling Technologies/New England Biolabs, Hitchin, UK).

Techniques: Transfection, Incubation, Expressing, Blocking Assay, Western Blot, Control, Extraction, Activity Assay, Kinase Assay

Journal: Nature communications

Article Title: Cell metabolism regulates integrin mechanosensing via an SLC3A2-dependent sphingolipid biosynthesis pathway.

doi: 10.1038/s41467-018-07268-w

Figure Lengend Snippet: Fig. 6 Depletion of DES2 recapitulates most of defects observed upon depletion of CD98hc. a Depletion of DES2 by siRNA impairs RhoA activation by mechanical force application on integrins. Right panel, quantification of RhoA activation, means are plotted with s.e.m. as error bars from n = 2 experiments, *P < 0.05 in a two-way ANOVA. b Depletion of DES2 by shRNA impairs SFK phosphorylation at Y416. One experiment representative of n = 2. c Depletion of DES2 impairs mechanically induced GEF-H1 activation. One experiment representative of n = 2. d Depletion of DES2 triggers GEF-H1 sequestration in cell membrane. s supernatant, p membrane pellet. One experiment representative of n = 2. e DES2 depletion partially inhibits mechanically coupled YAP/Taz activation as measured by RT-qPCR of ANKRD1 transcript. Means are plotted with s.e.m. as error bars from n = 6 with *P < 0.05 and ****P < 0.0001 in a two-way ANOVA. f Depletion of DES2 impairs generation of traction forces. Traction force microscopy was performed on subconfluent cells. Scale bar is 50 µm

Article Snippet: The rabbit monoclonal anti-GEF-H1 55B6 (1/500), anti-Src 36D10 (1/500), anti-Grp94 D6X2Q (1/500), anti-Grp78/BiP C50B12 (1/1000) and anti-Vimentin D21H3 (1/500) antibodies, and the rabbit polyclonal anti-phospho Y416 Src (1/500) and anti-flotillin-1 (1/1000) (#3253) antibodies were from Cell Signaling Technology.

Techniques: Activation Assay, shRNA, Phospho-proteomics, Membrane, Quantitative RT-PCR, Microscopy

Journal: Nature communications

Article Title: Cell metabolism regulates integrin mechanosensing via an SLC3A2-dependent sphingolipid biosynthesis pathway.

doi: 10.1038/s41467-018-07268-w

Figure Lengend Snippet: Fig. 7 Loss of CD98hc impairs GEF-H1and Src family kinase activation, and integrin dynamics. a Loss of CD98hc impairs LARG and GEF-H1 activation by mechanical force application on integrins. One experiment representative of n = 2. b Loss of CD98hc impairs Src activity but does not affect ERK1/2 phosphorylation by mechanical force application on integrins. Src activity was monitored by tracking substrate FAK Y576 phosphorylation and SFK phosphorylation at Y416. c Loss of CD98hc induces constitutive recruitment of GEF-H1 to the membrane. s supernatant, p membrane pellet. One experiment representative of n = 3. d Control or CD98hc-null dermal fibroblasts were stimulated with FN-coated magnetic beads. Cells were lysed and lysates were fractionated into cytosolic (s) and membrane (p) fractions by centrifugation. Active RhoGEFs was pulled-down from each fraction with GST- RhoA17A beads. One experiment representative of n = 2. e, f Diffusion rate (t-half) of integrin α5 and β3 as calculated from the curve fit generated from the FRAP measurements performed on n = 27, 41, and 67; and 73, 36, and 57 adhesions, respectively, for control, CD98hc-null and C330S cells on integrin α5 and β3. Error bars are 95% CI calculated from that fit. g, h Integrin α5 and β3 mobile fraction as calculated from the curve fit generated from the FRAP measurements. Error bars are 95 CI calculated from that fit. i Trafficking of integrin β1 in control or CD98hc-null cells. Integrin β1 was labeled with Alexa 488 coupled antibody then integrin trafficking was chased for indicated time. Extracellular staining was quenched and only intracellular labeled integrin is observed. Scale bar is 50 µm

Article Snippet: The rabbit monoclonal anti-GEF-H1 55B6 (1/500), anti-Src 36D10 (1/500), anti-Grp94 D6X2Q (1/500), anti-Grp78/BiP C50B12 (1/1000) and anti-Vimentin D21H3 (1/500) antibodies, and the rabbit polyclonal anti-phospho Y416 Src (1/500) and anti-flotillin-1 (1/1000) (#3253) antibodies were from Cell Signaling Technology.

Techniques: Activation Assay, Activity Assay, Phospho-proteomics, Membrane, Control, Magnetic Beads, Centrifugation, Diffusion-based Assay, Generated, Labeling, Staining