phosphorylated src family kinase y416  (Cell Signaling Technology Inc)

Journal: Cancer Science

Article Title: Lack of GD3 synthase (St8sia1) attenuates malignant properties of gliomas in genetically engineered mouse model

doi: 10.1111/cas.15032

Figure Lengend Snippet: Lack of GD3 synthase (GD3S) attenuated phosphorylation levels of signaling molecules in gliomas. A, Two weeks after 2 wk of injection of PDGFB‐T2A‐GFP cDNA‐expressing cells by replication‐competent avian leukemia virus splice acceptor, lysates of glioma or nonglioma brain tissues were prepared and applied to western blotting. Total proteins (50 μg) were analyzed using Abs, as indicated on the left side of individual images. Representative results of three sample sets including GD3S‐KO and control mice are shown. B‐E, Relative expression levels of GFP (B), relative phosphorylation levels of Src family kinases (SFKs) (C), Akt (D), and Erks (E) are plotted. GFP expressions and phosphorylation levels of SFKs were normalized by β‐actin bands. Phosphorylation levels of Akt and Erks were normalized by bands of total Akt and total Erks, respectively. Samples from seven GD3S‐KO mice and five control mice were analyzed. * P < .05, ** P < .01

Article Snippet: Other Abs for Akt, phosphorylated Akt (S473), Erks, phosphorylated Erks, and phosphorylated SFKs (Y417) were purchased from Cell Signaling Technology, and Abs for individual gangliosides were established in our laboratory.

Techniques: Phospho-proteomics, Injection, Expressing, Virus, Western Blot, Control

Journal: Cancer Science

Article Title: Lack of GD3 synthase (St8sia1) attenuates malignant properties of gliomas in genetically engineered mouse model

doi: 10.1111/cas.15032

Figure Lengend Snippet: Survival curves of GD3 synthase (GD3S)‐KO injected with GD3S expression vectors. A, Kaplan‐Meier analysis showing that GD3S‐KO mice injected with GD3S together with platelet‐derived growth factor B (PDGFB) expression vector (22 mice) tended to show a shorter survival time than GD3S‐KO mice ( P = .3436, log‐rank test). Other data such as simple transfection with PDGFB cDNA in GD3S‐KO and control mice were the same as those presented in Figure , showing longer survival of GD3S‐KO mice than WT mice. B, Summary of the conclusion of this study. Activation of Akt, Src family kinase (SFKs), and Erks and upregulation of MMP9 by Ap2α lead to malignant phenotypes of gliomas under expression of GD3/GD2 and/or GD3S. The mechanism of how GD3S induces strong expression of Ap2α remains to be investigated. PDGFRα, platelet‐derived growth factor receptor α

Article Snippet: Other Abs for Akt, phosphorylated Akt (S473), Erks, phosphorylated Erks, and phosphorylated SFKs (Y417) were purchased from Cell Signaling Technology, and Abs for individual gangliosides were established in our laboratory.

Techniques: Injection, Expressing, Derivative Assay, Plasmid Preparation, Transfection, Control, Activation Assay

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Western blotting (left panel) for total and phosphorylated SFK in the lungs from control IgG- and 38–2 mAb-treated mice at 3 dpi with 200 IFU of IAV/PR8. Actb, β-actin. The percent density of total SFK (middle panel) and SFK phosphorylated at tyrosine 416 (right panel) against the densities of the corresponding proteins in IgG-treated, infected lungs after being normalized by the densities of β-actin. **, p<0.01. ( B ) Survival rate (%, upper panels) and body weight loss (%, lower panels) of WT mice intraperitoneally administrated with control IgG (left panels) and 38–2 mAb (right panel) together with 20 mg/mouse of DS 1 day before intranasal infection with 200 IFU of IAV/PR8. Error bars, SD. ( C ) Western blotting (upper panel) for total SFK, phosphorylated SFK (Tyr416), the cleaved caspase 3, and the viral protein M2 in the lungs from control IgG/DMSO-, 38–2 mAb/DMSO-, control IgG/DS-, and 38–2 mAb/DS-treated mice at 3 dpi with 200 IFU of IAV/PR8. Actb, β-actin. Lungs from uninfected, saline/DMSO-treated mice were used as a control. The percent density (lower panels) of total SFK, phosphorylated SFK (Tyr416), the cleaved caspase 3, and the viral protein M2 against the densities of the corresponding proteins in uninfected, saline/DMSO-treated lungs after being normalized by the densities of β-actin. *, p<0.05; **, p<0.01.

Article Snippet: Anti-podoplanin antibody (Cat. No. D190-3) and anti-β-actin antibody (Cat. No. M177-3) from MBL (Nagoya, Japan;), anti-SP-C (Cat. No. sc-7706) and anti-CC10 antibodies (Cat. No. sc-9772) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pro-caspase 3 (Cat. No. 9662S), anti-cleaved caspase 3 (Cat. No. 9664S), anti-total SFK (Cat. No. 2109S) and anti-phosphorylated SFK (Tyr416) antibodies (Cat. No. 2101S) from Cell Signaling (Beverly, MA), anti-PB1 (Cat. No. GTX125923), anti-NS1 (Cat. No. GTX125990), anti-M2 (Cat. No. GTX125951) and anti-NP (Cat. No. GTX125989) antibodies from GeneTex (Irvine, CA), anti-SOD1 antibody (Cat. No. ab13498) from Abcam (Cambridge, UK), anti-CD3 (Cat. No. MAB4841), anti-MGL1/2 (Cat. No. AF4297) and HRP -conjugated anti-goat IgG antibodies (Cat. No. HAF017) from R&D systems (Minneapolis, MN), anti-MPO antibody from Proteintech (Rosemont, IL; Cat. No. 66177-1-Ig), PE anti-mouse/human CD11b antibody (Cat. No. 101207) from Biolegend (San Diego, CA), vitality full length hrGFP polyclonal antibody (Cat. No. 240141) from Agilent (Santa Clara, CA), HRP-conjugated anti-mouse IgG antibodies (Cat. No. NA931), HRP-conjugated anti-rabbit IgG antibodies (Cat. No. NA934), HRP-conjugated anti-rat IgG antibodies (Cat. No. NA935) from GE Healthcare (Buckinghamshire, UK), Alexa Fluor 596 donkey anti-mouse IgG antibody (Cat. No. A21203), Alexa Fluor 488 goat anti-rabbit IgG antibody (Cat. No. A11008), Alexa Fluor 596 donkey anti-goat IgG antibody (Cat. No. A11058), Texas Red-X goat anti-rat IgG antibody (Cat. No. T6392), and Alexa Fluor 647 goat anti-mouse antibody (Cat. No. A28181) from Invitrogen (Carlsbad, CA).

Techniques: Western Blot, Control, Infection, Saline

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Double immunofluorescent staining for phosphorylated SFK (Tyr416) together with MGL1/2, CD3, and MPO in the lungs from control IgG- and 38–2 mAb-treated mice uninfected and at 3 dpi with 200 IFU of IAV/PR8. Un, uninfected. Bar, 200 μ m. ( B ) Fold mRNA expression (2 -ΔCt ) analyzed by real-time RT-PCR for IL-6, TNF-α, IFN-α, INF-γ, MCP-1, iNOS, IFIT1, MxA, ARG1, MGL1, and IL-10 in the lungs from control IgG/DMSO, control IgG/DS-, 38–2 mAb/DMSO-, and 38–2 mAb/DS-treated mice uninfected and at 3 dpi with 200 IFU of IAV/PR8 (n = 3 in each group). Un, uninfected.*, p<0.05;**, p<0.01.

Article Snippet: Anti-podoplanin antibody (Cat. No. D190-3) and anti-β-actin antibody (Cat. No. M177-3) from MBL (Nagoya, Japan;), anti-SP-C (Cat. No. sc-7706) and anti-CC10 antibodies (Cat. No. sc-9772) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pro-caspase 3 (Cat. No. 9662S), anti-cleaved caspase 3 (Cat. No. 9664S), anti-total SFK (Cat. No. 2109S) and anti-phosphorylated SFK (Tyr416) antibodies (Cat. No. 2101S) from Cell Signaling (Beverly, MA), anti-PB1 (Cat. No. GTX125923), anti-NS1 (Cat. No. GTX125990), anti-M2 (Cat. No. GTX125951) and anti-NP (Cat. No. GTX125989) antibodies from GeneTex (Irvine, CA), anti-SOD1 antibody (Cat. No. ab13498) from Abcam (Cambridge, UK), anti-CD3 (Cat. No. MAB4841), anti-MGL1/2 (Cat. No. AF4297) and HRP -conjugated anti-goat IgG antibodies (Cat. No. HAF017) from R&D systems (Minneapolis, MN), anti-MPO antibody from Proteintech (Rosemont, IL; Cat. No. 66177-1-Ig), PE anti-mouse/human CD11b antibody (Cat. No. 101207) from Biolegend (San Diego, CA), vitality full length hrGFP polyclonal antibody (Cat. No. 240141) from Agilent (Santa Clara, CA), HRP-conjugated anti-mouse IgG antibodies (Cat. No. NA931), HRP-conjugated anti-rabbit IgG antibodies (Cat. No. NA934), HRP-conjugated anti-rat IgG antibodies (Cat. No. NA935) from GE Healthcare (Buckinghamshire, UK), Alexa Fluor 596 donkey anti-mouse IgG antibody (Cat. No. A21203), Alexa Fluor 488 goat anti-rabbit IgG antibody (Cat. No. A11008), Alexa Fluor 596 donkey anti-goat IgG antibody (Cat. No. A11058), Texas Red-X goat anti-rat IgG antibody (Cat. No. T6392), and Alexa Fluor 647 goat anti-mouse antibody (Cat. No. A28181) from Invitrogen (Carlsbad, CA).

Techniques: Staining, Control, Expressing, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Uncropped, full picture of Western blotting for PrP C with 38–2 mAb in the lungs (Lg) and peritoneal macrophages (PM) from WT and Prnp 0/0 mice. Actb, β-actin. ( B ) Western blotting for total SFK, phosphorylated SFK (Tyr416), and MGL1/2 in peritoneal macrophages from WT and Prnp 0/0 mice 3 hrs after treatment with control IgG and 38–2 mAb together with DS or control DMSO. ( C ) ELISA for INF-γ, TNF-α, IL-4, and IL-10 in the culture medium from WT peritoneal macrophages at 0.5, 4, 16, and 24 hours (hrs) after treatment with control IgG and 38–2 mAb.*, p<0.05;** p<0.01. ( D ) Western blotting for total SFK, phosphorylated SFK (Tyr416), and MGL1/2 in peritoneal macrophages (PM) from WT and Prnp 0/0 mice 1 and 3 days after peritoneal injection with control IgG and 38–2 mAb together with control DMSO and DS.

Article Snippet: Anti-podoplanin antibody (Cat. No. D190-3) and anti-β-actin antibody (Cat. No. M177-3) from MBL (Nagoya, Japan;), anti-SP-C (Cat. No. sc-7706) and anti-CC10 antibodies (Cat. No. sc-9772) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pro-caspase 3 (Cat. No. 9662S), anti-cleaved caspase 3 (Cat. No. 9664S), anti-total SFK (Cat. No. 2109S) and anti-phosphorylated SFK (Tyr416) antibodies (Cat. No. 2101S) from Cell Signaling (Beverly, MA), anti-PB1 (Cat. No. GTX125923), anti-NS1 (Cat. No. GTX125990), anti-M2 (Cat. No. GTX125951) and anti-NP (Cat. No. GTX125989) antibodies from GeneTex (Irvine, CA), anti-SOD1 antibody (Cat. No. ab13498) from Abcam (Cambridge, UK), anti-CD3 (Cat. No. MAB4841), anti-MGL1/2 (Cat. No. AF4297) and HRP -conjugated anti-goat IgG antibodies (Cat. No. HAF017) from R&D systems (Minneapolis, MN), anti-MPO antibody from Proteintech (Rosemont, IL; Cat. No. 66177-1-Ig), PE anti-mouse/human CD11b antibody (Cat. No. 101207) from Biolegend (San Diego, CA), vitality full length hrGFP polyclonal antibody (Cat. No. 240141) from Agilent (Santa Clara, CA), HRP-conjugated anti-mouse IgG antibodies (Cat. No. NA931), HRP-conjugated anti-rabbit IgG antibodies (Cat. No. NA934), HRP-conjugated anti-rat IgG antibodies (Cat. No. NA935) from GE Healthcare (Buckinghamshire, UK), Alexa Fluor 596 donkey anti-mouse IgG antibody (Cat. No. A21203), Alexa Fluor 488 goat anti-rabbit IgG antibody (Cat. No. A11008), Alexa Fluor 596 donkey anti-goat IgG antibody (Cat. No. A11058), Texas Red-X goat anti-rat IgG antibody (Cat. No. T6392), and Alexa Fluor 647 goat anti-mouse antibody (Cat. No. A28181) from Invitrogen (Carlsbad, CA).

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Injection

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Western blotting for total SFK, phosphorylated SFK (Tyr416), and MGL1/2 in peritoneal macrophages from WT and Prnp 0/0 mice 3 hrs after treatment with control IgG and 3S9 and 2H9 mAbs together with control DMSO and DS. ( B ) Survival rate (%, upper panels) and body weight loss (%, lower panels) of WT mice intraperitoneally administered with control IgG and 3S9 and 2H9 mAbs 1 day before intranasal infection with 100 (left panels) or 200 IFU (right panels) of IAV/PR8. Error bars, SD. *, p<0.05; **, p<0.01; IgG vs 3S9. #, <0.05; IgG vs 2H9.

Article Snippet: Anti-podoplanin antibody (Cat. No. D190-3) and anti-β-actin antibody (Cat. No. M177-3) from MBL (Nagoya, Japan;), anti-SP-C (Cat. No. sc-7706) and anti-CC10 antibodies (Cat. No. sc-9772) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pro-caspase 3 (Cat. No. 9662S), anti-cleaved caspase 3 (Cat. No. 9664S), anti-total SFK (Cat. No. 2109S) and anti-phosphorylated SFK (Tyr416) antibodies (Cat. No. 2101S) from Cell Signaling (Beverly, MA), anti-PB1 (Cat. No. GTX125923), anti-NS1 (Cat. No. GTX125990), anti-M2 (Cat. No. GTX125951) and anti-NP (Cat. No. GTX125989) antibodies from GeneTex (Irvine, CA), anti-SOD1 antibody (Cat. No. ab13498) from Abcam (Cambridge, UK), anti-CD3 (Cat. No. MAB4841), anti-MGL1/2 (Cat. No. AF4297) and HRP -conjugated anti-goat IgG antibodies (Cat. No. HAF017) from R&D systems (Minneapolis, MN), anti-MPO antibody from Proteintech (Rosemont, IL; Cat. No. 66177-1-Ig), PE anti-mouse/human CD11b antibody (Cat. No. 101207) from Biolegend (San Diego, CA), vitality full length hrGFP polyclonal antibody (Cat. No. 240141) from Agilent (Santa Clara, CA), HRP-conjugated anti-mouse IgG antibodies (Cat. No. NA931), HRP-conjugated anti-rabbit IgG antibodies (Cat. No. NA934), HRP-conjugated anti-rat IgG antibodies (Cat. No. NA935) from GE Healthcare (Buckinghamshire, UK), Alexa Fluor 596 donkey anti-mouse IgG antibody (Cat. No. A21203), Alexa Fluor 488 goat anti-rabbit IgG antibody (Cat. No. A11008), Alexa Fluor 596 donkey anti-goat IgG antibody (Cat. No. A11058), Texas Red-X goat anti-rat IgG antibody (Cat. No. T6392), and Alexa Fluor 647 goat anti-mouse antibody (Cat. No. A28181) from Invitrogen (Carlsbad, CA).

Techniques: Western Blot, Control, Infection

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Western blotting (left panel) for total and phosphorylated SFK in the lungs from control IgG- and 38–2 mAb-treated mice at 3 dpi with 200 IFU of IAV/PR8. Actb, β-actin. The percent density of total SFK (middle panel) and SFK phosphorylated at tyrosine 416 (right panel) against the densities of the corresponding proteins in IgG-treated, infected lungs after being normalized by the densities of β-actin. **, p<0.01. ( B ) Survival rate (%, upper panels) and body weight loss (%, lower panels) of WT mice intraperitoneally administrated with control IgG (left panels) and 38–2 mAb (right panel) together with 20 mg/mouse of DS 1 day before intranasal infection with 200 IFU of IAV/PR8. Error bars, SD. ( C ) Western blotting (upper panel) for total SFK, phosphorylated SFK (Tyr416), the cleaved caspase 3, and the viral protein M2 in the lungs from control IgG/DMSO-, 38–2 mAb/DMSO-, control IgG/DS-, and 38–2 mAb/DS-treated mice at 3 dpi with 200 IFU of IAV/PR8. Actb, β-actin. Lungs from uninfected, saline/DMSO-treated mice were used as a control. The percent density (lower panels) of total SFK, phosphorylated SFK (Tyr416), the cleaved caspase 3, and the viral protein M2 against the densities of the corresponding proteins in uninfected, saline/DMSO-treated lungs after being normalized by the densities of β-actin. *, p<0.05; **, p<0.01.

Article Snippet: Cells were lysed in RIPA lysis buffer (1% NP-40, 0.25% Sodium deoxycholate, 0.05% SDS, 150 mM NaCl, 20 mM HEPES-NaOH, pH 7.5, 1 mM EGTA, and 1 mM MgCl 2 ) containing Complete protease inhibitors (Merck Millipore, Darmstadt, Germany), PhosSTOP phosphatase inhibitors (Merck Millipore), and 50 units/ml of benzonase endonuclease (Merck Millipore) on ice for 10 min. After centrifugation at 20,000×g for 15 min at 4°C, the supernatants were incubated with anti-phosphorylated SFK (Tyr416) antibody (Cell Signaling Technology) that was immobilized on SureBeads Protein G magnetic beads (Bio-Rad, Hercules, CA) for 3 hrs at 4°C with rotation.

Techniques: Western Blot, Control, Infection, Saline

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Double immunofluorescent staining for phosphorylated SFK (Tyr416) together with MGL1/2, CD3, and MPO in the lungs from control IgG- and 38–2 mAb-treated mice uninfected and at 3 dpi with 200 IFU of IAV/PR8. Un, uninfected. Bar, 200 μ m. ( B ) Fold mRNA expression (2 -ΔCt ) analyzed by real-time RT-PCR for IL-6, TNF-α, IFN-α, INF-γ, MCP-1, iNOS, IFIT1, MxA, ARG1, MGL1, and IL-10 in the lungs from control IgG/DMSO, control IgG/DS-, 38–2 mAb/DMSO-, and 38–2 mAb/DS-treated mice uninfected and at 3 dpi with 200 IFU of IAV/PR8 (n = 3 in each group). Un, uninfected.*, p<0.05;**, p<0.01.

Article Snippet: Cells were lysed in RIPA lysis buffer (1% NP-40, 0.25% Sodium deoxycholate, 0.05% SDS, 150 mM NaCl, 20 mM HEPES-NaOH, pH 7.5, 1 mM EGTA, and 1 mM MgCl 2 ) containing Complete protease inhibitors (Merck Millipore, Darmstadt, Germany), PhosSTOP phosphatase inhibitors (Merck Millipore), and 50 units/ml of benzonase endonuclease (Merck Millipore) on ice for 10 min. After centrifugation at 20,000×g for 15 min at 4°C, the supernatants were incubated with anti-phosphorylated SFK (Tyr416) antibody (Cell Signaling Technology) that was immobilized on SureBeads Protein G magnetic beads (Bio-Rad, Hercules, CA) for 3 hrs at 4°C with rotation.

Techniques: Staining, Control, Expressing, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Uncropped, full picture of Western blotting for PrP C with 38–2 mAb in the lungs (Lg) and peritoneal macrophages (PM) from WT and Prnp 0/0 mice. Actb, β-actin. ( B ) Western blotting for total SFK, phosphorylated SFK (Tyr416), and MGL1/2 in peritoneal macrophages from WT and Prnp 0/0 mice 3 hrs after treatment with control IgG and 38–2 mAb together with DS or control DMSO. ( C ) ELISA for INF-γ, TNF-α, IL-4, and IL-10 in the culture medium from WT peritoneal macrophages at 0.5, 4, 16, and 24 hours (hrs) after treatment with control IgG and 38–2 mAb.*, p<0.05;** p<0.01. ( D ) Western blotting for total SFK, phosphorylated SFK (Tyr416), and MGL1/2 in peritoneal macrophages (PM) from WT and Prnp 0/0 mice 1 and 3 days after peritoneal injection with control IgG and 38–2 mAb together with control DMSO and DS.

Article Snippet: Cells were lysed in RIPA lysis buffer (1% NP-40, 0.25% Sodium deoxycholate, 0.05% SDS, 150 mM NaCl, 20 mM HEPES-NaOH, pH 7.5, 1 mM EGTA, and 1 mM MgCl 2 ) containing Complete protease inhibitors (Merck Millipore, Darmstadt, Germany), PhosSTOP phosphatase inhibitors (Merck Millipore), and 50 units/ml of benzonase endonuclease (Merck Millipore) on ice for 10 min. After centrifugation at 20,000×g for 15 min at 4°C, the supernatants were incubated with anti-phosphorylated SFK (Tyr416) antibody (Cell Signaling Technology) that was immobilized on SureBeads Protein G magnetic beads (Bio-Rad, Hercules, CA) for 3 hrs at 4°C with rotation.

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Injection

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Western blotting for total SFK, phosphorylated SFK (Tyr416), and MGL1/2 in peritoneal macrophages from WT and Prnp 0/0 mice 3 hrs after treatment with control IgG and 3S9 and 2H9 mAbs together with control DMSO and DS. ( B ) Survival rate (%, upper panels) and body weight loss (%, lower panels) of WT mice intraperitoneally administered with control IgG and 3S9 and 2H9 mAbs 1 day before intranasal infection with 100 (left panels) or 200 IFU (right panels) of IAV/PR8. Error bars, SD. *, p<0.05; **, p<0.01; IgG vs 3S9. #, <0.05; IgG vs 2H9.

Article Snippet: Cells were lysed in RIPA lysis buffer (1% NP-40, 0.25% Sodium deoxycholate, 0.05% SDS, 150 mM NaCl, 20 mM HEPES-NaOH, pH 7.5, 1 mM EGTA, and 1 mM MgCl 2 ) containing Complete protease inhibitors (Merck Millipore, Darmstadt, Germany), PhosSTOP phosphatase inhibitors (Merck Millipore), and 50 units/ml of benzonase endonuclease (Merck Millipore) on ice for 10 min. After centrifugation at 20,000×g for 15 min at 4°C, the supernatants were incubated with anti-phosphorylated SFK (Tyr416) antibody (Cell Signaling Technology) that was immobilized on SureBeads Protein G magnetic beads (Bio-Rad, Hercules, CA) for 3 hrs at 4°C with rotation.

Techniques: Western Blot, Control, Infection