Journal: Microbiology Spectrum

Article Title: Pseudomonas aeruginosa Induces Interferon-β Production to Promote Intracellular Survival

doi: 10.1128/spectrum.01550-22

Figure Lengend Snippet: The activated NLRP3 and NLRC4 inflammasomes inhibit the production of IFN-β. (A) The mRNA relative expression of IL-1β in immortalized bone marrow-derived macrophages (iBMDMs) with PA (MOI = 10) infection for 1 h ( n ≥ 3). (B) Immunoblotting for pro-interleukin-1β (pro-IL-1β), IL-1β, and GAPDH of iBMDM with PA (MOI = 5/10) infection for indicated time; the level of IL-1β in mouse peritoneal macrophages supernatant for PA (MOI = 5) infection for 2 h ( n ≥ 3). (C) The level of IL-1β in WT and NLRP3 KO mouse peritoneal macrophages supernatant for PA (MOI = 1) infection for indicated time ( n ≥ 3). (D) The level of IL-1β in WT and NLRC4 KO mouse peritoneal macrophages supernatant for PA (MOI = 1) infection for indicated time ( n ≥ 3). (E) The mRNA relative expression of IFN-β in WT and NLRP3 KO mouse peritoneal macrophages with PA (MOI = 1) infection for indicated time ( n ≥ 3). (F) The mRNA relative expression of IFN-β in WT and NLRC4 KO mouse peritoneal macrophages with PA (MOI = 1) infection for indicated time ( n ≥ 3). (G) The mRNA relative expression of IFN-β in WT and NLRP3 KO mouse lung with PA (3 × 10 6 CFU) infection for 24 h ( n ≥ 3). (H) The mRNA relative expression of IFN-β in WT and NLRC4 KO mouse lung with PA (3 × 10 6 CFU) infection for 24 h ( n ≥ 3). (I) Pretreatment with Nig (5 μM) or DMSO as a control for 2 h; the mRNA relative expression of IFN-β in mouse peritoneal macrophages with PA (MOI = 1) infection for 3 h ( n ≥ 3), pretreatment with Nig (5 μM) or DMSO as a control for 2 h, and the level of IFN-β of the mouse peritoneal macrophages supernatant with PA (MOI = 1) infection for 1 h ( n ≥ 3). (J) Pretreatment with Nig (5 μM) or DMSO as a control for 2 h, immunoblotting for total TBK1, P-TBK1, and GAPDH in mouse peritoneal macrophages with PA (MOI = 5) infection for 1 h ( n ≥ 3). (K) Pretreatment with VX-765 (10 μM) or DMSO as a control for 3 h, the mRNA expression of IFN-β in mouse peritoneal macrophages, and the level of macrophages supernatant with PA (MOI = 1) infection for 2 h ( n ≥ 3). All experiments were performed three times. * , P < 0.05; * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001; LPS, lipopolysaccharide; Nig, nigericin.

Article Snippet: The cells were pretreated with VX-765 (HY-13205, Med Chem Express), Nig (HY-13205, Med Chem Express), IL-1β (HY-P7073, Med Chem Express), IL-1β receptor inhibitor (TLR-1, HY-W011400, Med Chem Express), AKT inhibitor VIII (HY-10355, Med Chem Express), or IFN-β (CAT 300-02BC, PeproTech) for the indicated times before stimulation with PA, as indicated; multiplicity of infection (MOI) values are given for the different times.

Techniques: Expressing, Derivative Assay, Infection, Western Blot, Control

Journal: Microbiology Spectrum

Article Title: Pseudomonas aeruginosa Induces Interferon-β Production to Promote Intracellular Survival

doi: 10.1128/spectrum.01550-22

Figure Lengend Snippet: IL-1β downstream of NLRP3 and NLRC4 inflammasome suppressed IFN-β production. (A) Pretreatment with TLR-1 (33 μM) or ethanol as a control for 3 h; the mRNA expression of IFN-β and CXCL-10 in iBMDM with PA (MOI = 1) infection for 3 h ( n ≥ 3). (B) Pretreatment with TLR-1 (33 μM) or ethanol as a control for 3 h; the level of IFN-β in iBMDM supernatant for PA (MOI = 1) infection for 3 h ( n ≥ 3). (C) Pretreatment with TLR-1 (33 μM) or ethanol as a control for 3 h; immunoblotting for total TBK1, P-TBK1, total IRF3, and P-IRF3 and GAPDH in iBMDM with PA (MOI = 2) infection for 1 h. (D) Pretreatment with TLR-1 (33 μM) or ethanol as a control for 3 h; the mRNA expression of IFN-β, CXCL-10, and CCL-5 in mouse peritoneal macrophages with PA (MOI = 1) infection for 3 h ( n ≥ 3). (E) Pretreatment with IL-1β (10 ng/mL) or PBS as a control for 3 h; the mRNA relative expression of IFN-β in mouse peritoneal macrophages with PA (MOI = 1) infection for 1 h ( n ≥ 3). (F) Pretreatment with IL-1β (10 ng/mL) or PBS as a control for 3 h; the mRNA relative expression of IL-1β in mouse peritoneal macrophages with PA (MOI = 1) infection for 1 h ( n ≥ 3). (G) Pretreatment with IL-1β (10 ng/mL) or PBS as a control for 3 h; the level of IFN-β in mouse peritoneal macrophages supernatant with PA (MOI = 1) infection for 1 h ( n ≥ 3). (H) Pretreatment with IL-1β (20 ng/mL) or PBS as a control for 3 h; immunoblotting for total TBK1 and P-TBK1 and GAPDH in MH-S cells with PA (MOI = 2) infection for 1 h. All experiments were performed three times. * , P < 0.05; * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001.

Article Snippet: The cells were pretreated with VX-765 (HY-13205, Med Chem Express), Nig (HY-13205, Med Chem Express), IL-1β (HY-P7073, Med Chem Express), IL-1β receptor inhibitor (TLR-1, HY-W011400, Med Chem Express), AKT inhibitor VIII (HY-10355, Med Chem Express), or IFN-β (CAT 300-02BC, PeproTech) for the indicated times before stimulation with PA, as indicated; multiplicity of infection (MOI) values are given for the different times.

Techniques: Control, Expressing, Infection, Western Blot

Journal: Microbiology Spectrum

Article Title: Pseudomonas aeruginosa Induces Interferon-β Production to Promote Intracellular Survival

doi: 10.1128/spectrum.01550-22

Figure Lengend Snippet: IL-1β inhibited cGAS-STING-TBK1-IFN-β axis by reducing the production of cGAMP. (A) Transfection with siRNA cGAS, immunoblotting for total TBK1, P-TBK1, total IRF3, cGAS, and GAPDH of MH-S with PA (MOI = 2) infection for 1 h. (B) Transfection with siRNA STING; immunoblotting for total TBK1, P-TBK1, total IRF3, STING, and GAPDH of MH-S with PA (MOI = 2) infection for 1 h. (C) Pretreatment with IL-1β (20 ng/mL) or PBS as a control for 3 h; the level of cGAMP in RAW264.7cells with PA (MOI = 5) infection for 45 min ( n ≥ 3). (D) Pretreatment with TLR-1 (33 μM) or ethanol as a control for 3 h; the level of cGAMP in RAW264.7cells with PA (MOI = 5) infection for 45 min ( n ≥ 3). (E) Pretreatment with AKT inhibitor (10 μM) or DMSO as a control for 3 h; the mRNA relative expression of IFN-β in RAW264.7 cells with PA (MOI = 5) infection for indicated time ( n ≥ 3). (F) Pretreatment with AKT inhibitor (10 μM) or DMSO as a control for 3 h; immunoblotting for total AKT, P-AKT, total TBK1, P-TBK1, and GAPDH in RAW264.7 cells with PA (MOI = 5) infection for 45 min. (G) Pretreatment with IL-1β (20 ng/mL) or PBS as a control for 3 h; immunoblotting for total AKT, P-AKT, and GAPDH in RAW264.7 cells with PA (MOI = 5) infection for 45 min. (H) Pretreatment with IL-1β (20 ng/mL) and AKT inhibitor (10 μM) or PBS and DMSO as a control for 3 h; immunoblotting for total AKT, P-AKT, total TBK1, P-TBK1, and GAPDH in RAW264.7 cells with PA (MOI = 5) infection for 45 min. (I) Pretreatment with IL-1β (20 ng/mL) and AKT inhibitor (10 μM) or PBS and DMSO as a control for 3 h; the level of cGAMP in RAW264.7 cells with PA (MOI = 5) infection for 1 h ( n ≥ 3). (J) Pretreatment with IL-1β (20 ng/mL) and AKT inhibitor (10 μM) or PBS and DMSO as a control for 3 h; the mRNA relative expression of IFN-β in RAW264.7 cells with PA (MOI = 5) infection for 1 h ( n ≥ 3). All experiments were performed three times. * , P < 0.05; * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001.

Article Snippet: The cells were pretreated with VX-765 (HY-13205, Med Chem Express), Nig (HY-13205, Med Chem Express), IL-1β (HY-P7073, Med Chem Express), IL-1β receptor inhibitor (TLR-1, HY-W011400, Med Chem Express), AKT inhibitor VIII (HY-10355, Med Chem Express), or IFN-β (CAT 300-02BC, PeproTech) for the indicated times before stimulation with PA, as indicated; multiplicity of infection (MOI) values are given for the different times.

Techniques: Transfection, Western Blot, Infection, Control, Expressing

Journal: Microbiology Spectrum

Article Title: Pseudomonas aeruginosa Induces Interferon-β Production to Promote Intracellular Survival

doi: 10.1128/spectrum.01550-22

Figure Lengend Snippet: IFN-β promoted the survival of intracellular PA in macrophages. (A) The percentage of intracellular PA of WT and cGAS KO mouse peritoneal macrophages with PA (MOI = 0.1) infection for 2 h ( n ≥ 3). (B) The percentage of intracellular PA of WT and STING KO mouse peritoneal macrophages with PA (MOI = 0.1) infection for 2 h ( n ≥ 3). (C) Pretreatment with Nig (5 μM) or DMSO as a control for 2 h; the percentage of intracellular PA of mouse peritoneal macrophages with PA (MOI = 0.1) infection for 1 h ( n ≥ 3). (D) Pretreatment with VX-765 (10 μM) or DMSO as a control for 3 h; the percentage of intracellular PA of mouse peritoneal macrophages with PA (MOI = 0.1) infection for 1 h ( n ≥ 3). (E) The percentage of intracellular PA in WT, NLRP3 KO, and NLRC4 KO mouse peritoneal macrophages with PA (MOI = 0.1) infection for 1 h ( n ≥ 3). (F) Pretreatment with IL-1β (10 ng/mL) or ethanol as a control for 3 h; the percentage of intracellular PA in RAW264.7 cells with PA (MOI = 0.1) infection for 1 h ( n ≥ 3). (G) Pretreatment with TLR-1 (33 μM) or ethanol as a control for 3 h; the percentage of intracellular PA in RAW264.7 cells with PA (MOI = 0.1) infection for 1 h ( n ≥ 3). (H) Pretreatment with TLR-1 (33 μM) or ethanol as a control for 3 h; the intracellular PA in RAW264.7 cells with GFP-labeled PA (MOI = 5) infection for 2 h. (I) Representation appearance of lungs from WT and IFN-β KO mice with PA (3 × 10 6 CFU) infection for 24 h. Hematoxylin and eosin (H&E) staining of lungs from WT and IFN-β KO mice with PA (3 × 10 6 CFU) infection for 24 h. (J) The lung injury score of WT and IFN-β KO mice right lung with PA (3 × 10 6 CFU) infection for 24 h ( n ≥ 3). (K) The bacterial CFU of WT and IFN-β KO mice right lung with PA (3 × 10 6 CFU) infection for 24 h ( n ≥ 3). All experiments were performed three times. * , P < 0.05; * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001.

Article Snippet: The cells were pretreated with VX-765 (HY-13205, Med Chem Express), Nig (HY-13205, Med Chem Express), IL-1β (HY-P7073, Med Chem Express), IL-1β receptor inhibitor (TLR-1, HY-W011400, Med Chem Express), AKT inhibitor VIII (HY-10355, Med Chem Express), or IFN-β (CAT 300-02BC, PeproTech) for the indicated times before stimulation with PA, as indicated; multiplicity of infection (MOI) values are given for the different times.

Techniques: Infection, Control, Labeling, Staining

Journal: Microbiology Spectrum

Article Title: Pseudomonas aeruginosa Induces Interferon-β Production to Promote Intracellular Survival

doi: 10.1128/spectrum.01550-22

Figure Lengend Snippet: Graphical abstract of the research. PA triggered IFN-β production to promote its intracellular through cGAS-STING-TBK1 pathway. For host defense, IL-1β secreted by macrophages induced by PA suppress IFN-β response driven by cGAS-STING-TBK1 pathway. Mechanistically, IL-1β promoted AKT phosphorylation by binding to IL-1 receptors on the cell membrane. Phosphorylated AKT reduced the production of GAMP and inhibited the cGAS-STING-TBK1 signaling pathway, resulting in a decrease in the production of IFN-β. cGAMP, cyclic GMP-AMP; cGAS, cGAMP synthase.

Article Snippet: The cells were pretreated with VX-765 (HY-13205, Med Chem Express), Nig (HY-13205, Med Chem Express), IL-1β (HY-P7073, Med Chem Express), IL-1β receptor inhibitor (TLR-1, HY-W011400, Med Chem Express), AKT inhibitor VIII (HY-10355, Med Chem Express), or IFN-β (CAT 300-02BC, PeproTech) for the indicated times before stimulation with PA, as indicated; multiplicity of infection (MOI) values are given for the different times.

Techniques: Phospho-proteomics, Binding Assay, Membrane

Journal: Infection and Immunity

Article Title: Essential role of Helicobacter pylori apolipoprotein N-acyltransferase (Lnt) in stomach colonization

doi: 10.1128/iai.00369-23

Figure Lengend Snippet: Characterization of purified Lpp20 as model lipoprotein. (A) Lpp20-HA was immunopurified from WT or lnt mutant H. pylori strains VM396 and VM404. A representative Coomassie-stained gel is shown (lane a, WT Lpp20-HA from strain VM396; lane b, Δlnt Lpp20-HA from strain VM404). (B) Purified Lpp20-HA (0.1 µg/mL from either WT or lnt mutant H. pylori) was incubated with secreted embryonic alkaline phosphatase (SEAP) reporter cell lines HEK-Blue Null2 (Null), HEK-Blue hTLR4 (TLR4), HEK-Blue hTLR2 KO-TLR1/6 (KO 1/6), HEK-Blue hTLR2-TLR1 (TLR2/1), and HEK-Blue hTLR2-TLR6 (TLR2/6) from InvivoGen. Relative amounts of SEAP were measured after 16-h treatment. Results show the mean and standard deviation from single Lpp20-HA preparations (from both WT and lnt mutant H. pylori) tested in triplicate and are representative of three independent preparations of each lipoprotein. (C) Synthetic lipopeptides (0.04 µg/mL triacylated Pam3-CSK4 and diacylated Pam2-CSK4) were used to stimulate reporter cell lines as described in B. Results show the mean and standard deviation of triplicate determinations and are representative of three independent experiments. Results in panels B and C were analyzed by analysis of variance followed by Tukey’s post hoc test. Select pair-wise comparisons are indicated as significantly different (*, P < 0.001) or not significantly different (ns).

Article Snippet: A representative Coomassie-stained gel is shown (lane a, WT Lpp20-HA from strain VM396; lane b, Δ lnt Lpp20-HA from strain VM404). ( B ) Purified Lpp20-HA (0.1 µg/mL from either WT or lnt mutant H. pylori ) was incubated with secreted embryonic alkaline phosphatase (SEAP) reporter cell lines HEK-Blue Null2 (Null), HEK-Blue hTLR4 (TLR4), HEK-Blue hTLR2 KO-TLR1/6 (KO 1/6), HEK-Blue hTLR2-TLR1 (TLR2/1), and HEK-Blue hTLR2-TLR6 (TLR2/6) from InvivoGen.

Techniques: Purification, Mutagenesis, Staining, Incubation, Standard Deviation

Journal: Infection and Immunity

Article Title: Essential role of Helicobacter pylori apolipoprotein N-acyltransferase (Lnt) in stomach colonization

doi: 10.1128/iai.00369-23

Figure Lengend Snippet: Colonization of mice. (A) Wild-type C57Bl/6 were infected orogastrically with two doses of H. pylori J166 wild type, Δlnt mutant strain VM391, or the complemented mutant strain VM392 (Δlnt/lnt) as indicated. Colony forming units (CFUs) were determined 2 weeks post infection by plating serial dilutions of stomach homogenates. Data are representative of one of three independent experiments. (B) Tlr2−/− mice were infected orogastrically with two doses of H. pylori J166 wild type or Δlnt mutant strain VM391 as indicated. CFU were determined 2 weeks post infection by plating serial dilutions of stomach homogenates.

Article Snippet: A representative Coomassie-stained gel is shown (lane a, WT Lpp20-HA from strain VM396; lane b, Δ lnt Lpp20-HA from strain VM404). ( B ) Purified Lpp20-HA (0.1 µg/mL from either WT or lnt mutant H. pylori ) was incubated with secreted embryonic alkaline phosphatase (SEAP) reporter cell lines HEK-Blue Null2 (Null), HEK-Blue hTLR4 (TLR4), HEK-Blue hTLR2 KO-TLR1/6 (KO 1/6), HEK-Blue hTLR2-TLR1 (TLR2/1), and HEK-Blue hTLR2-TLR6 (TLR2/6) from InvivoGen.

Techniques: Infection, Mutagenesis

Journal: Infection and Immunity

Article Title: Essential role of Helicobacter pylori apolipoprotein N-acyltransferase (Lnt) in stomach colonization

doi: 10.1128/iai.00369-23

Figure Lengend Snippet: Diacylated lipoproteins vs control

Article Snippet: A representative Coomassie-stained gel is shown (lane a, WT Lpp20-HA from strain VM396; lane b, Δ lnt Lpp20-HA from strain VM404). ( B ) Purified Lpp20-HA (0.1 µg/mL from either WT or lnt mutant H. pylori ) was incubated with secreted embryonic alkaline phosphatase (SEAP) reporter cell lines HEK-Blue Null2 (Null), HEK-Blue hTLR4 (TLR4), HEK-Blue hTLR2 KO-TLR1/6 (KO 1/6), HEK-Blue hTLR2-TLR1 (TLR2/1), and HEK-Blue hTLR2-TLR6 (TLR2/6) from InvivoGen.

Techniques: Membrane, Ligand Binding Assay

Journal: Journal of leukocyte biology

Article Title: Susceptibility to tuberculosis is associated with TLR1 polymorphisms resulting in a lack of TLR1 cell surface expression.

doi: 10.1189/jlb.0409233

Figure Lengend Snippet: Figure 1. TLR1 deficiency is found on all leukocyte subpopulations. Granulocyte, monocyte, and lymphocyte populations of peripheral blood sam- ples of a TLR1neg (lower panel) and a TLR1pos (upper panel) individual were stained with anti-TLR1 antibodies and analyzed by flow cytometry. The fluorescence intensity of TLR1 (black) and the IgG1 isotype control (gray) is shown for all leukocyte populations. The results are representa- tive for 29 tested blood samples from healthy individuals.

Article Snippet: Blots were incubated with 1.5 g/ml biotylated anti-human TLR1 polyclonal antibodies (R&D Systems, Wiesbaden, Germany).

Techniques: Staining, Cytometry, Control

Journal: Journal of leukocyte biology

Article Title: Susceptibility to tuberculosis is associated with TLR1 polymorphisms resulting in a lack of TLR1 cell surface expression.

doi: 10.1189/jlb.0409233

Figure Lengend Snippet: Figure 2. TLR1 and CD14 surface expression is only up-regulated on TLR1pos granulocytes. (A) Stimulation of whole blood from TLR1pos (n3) and TLR1neg (n4) donors with 250 ng/ml LPS or 50 U/ml GM-CSF or medium for 90 min, 3 h, and 22 h. For 90-min values, only two donors were examined. The samples were stained indirectly with isotypic mAb controls and TLR1 mAb and analyzed by flow cytometry gated on granulocytes. Significant differences in the up-regulation of TLR1 antigen between TLR1neg blood granulocytes (black bars) and

Article Snippet: Blots were incubated with 1.5 g/ml biotylated anti-human TLR1 polyclonal antibodies (R&D Systems, Wiesbaden, Germany).

Techniques: Expressing, Staining, Cytometry

Journal: Journal of leukocyte biology

Article Title: Susceptibility to tuberculosis is associated with TLR1 polymorphisms resulting in a lack of TLR1 cell surface expression.

doi: 10.1189/jlb.0409233

Figure Lengend Snippet: Figure 4. Effects of mycobacteria, TLR1, TLR4, and TLR6 agonists on the production of TNF- by TLR1neg and TLR1pos PBMCs. (A) PBMCs from TLR1neg (black bars, n3) and TLR1pos (white bars, n3) donors were incubated with or without 50 ng/ml Pam3CSK4 or M. fortuitum, and superna- tants were harvested after 4 h. TNF- production was determined by ELISA. (B) PBMCs isolated from TLR1neg (black bars, n6) and TLR1pos (white bars, n6) donors were infected with or without M. bovis BCG or M. fortui- tum for 4 h. TNF- secretion was determined by ELISA. (C) PBMCs isolated from TLR1neg (●, n6) and TLR1pos (, n6) donors were not stimu- lated and stimulated with 10 ng/ml synthetic diacylated lipopeptide FSL-1, LPS, and Pam3CSK4, respectively. After 4 h, supernatants were collected, and TNF- release was determined by ELISA. For stimulation experiments with LPS, only three TLR1neg and three TLR1pos donors were analyzed. Median values (black horizontal lines) and log of observed data are depicted.

Article Snippet: Blots were incubated with 1.5 g/ml biotylated anti-human TLR1 polyclonal antibodies (R&D Systems, Wiesbaden, Germany).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Isolation, Infection

Journal: Science advances

Article Title: Injectable hydrogel with MSNs/microRNA-21-5p delivery enables both immunomodification and enhanced angiogenesis for myocardial infarction therapy in pigs.

doi: 10.1126/sciadv.abd6740

Figure Lengend Snippet: Fig. 2. The mechanism underlying the effects of the MSN complex on modifying the immune response. (A) A heatmap of selected proteins representing major altered signaling pathways in three datasets of macrophages treated with MSNs, MSN/miR-NC, or MSN/miR-21-5p complexes. Macrophages with no treatment were used as a negative control. The color bar indicates normalized z score intensity-based absolute quantification. (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of both up- and down-regulated pathways in macrophages after MSN treatment. The most significant pathways in the phosphoproteome are plotted on the x axis as the −log10 of the P value, compared with the proteome. (C) KEGG pathway map of Toll-like signaling pathway. Proteins shown with red backgrounds are down-regulated in macrophages after MSN complex treatments when compared with macrophages with no treatment, as determined by pathway analysis. (D) Real-time quantitative PCR and Western blot analysis of TLR1, TLR2, TLR3, TLR8, P-NFB, TNF-, IL-1, and IL-6 protein content alteration in macrophages after treatment with MSNs, MSN/miR-NC, or MSN/miR-21-5p complexes. (E) Real-time quantitative PCR and Western blot analysis of P-NFB, TNF-, IL-1, and IL-6 protein content alteration in MSN/miR-21-5p complex–treated macrophages that overexpress TLR2 with the TLR2 overexpression vector. ***P < 0.01. n = 3 per group. The data are shown as means ± SD. Photo credit: Yan Li, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

Article Snippet: The membranes were blocked with 5% bovine serum albumin and incubated with specific primary antibodies against the following: TNF- (AF-410-NA, R&D, USA), IL-1 (Novus, AF-401-NA), IL-6 (bs-0782R, Bioss, USA), VEGFA (DF7470, Affinity, USA), PDGFBB (bs-1316R, Bioss), TLR1 (NB100-56563, Novus), TLR2 (Abcam, ab209217), TLR3 (NBP2-24875, Novus), TLR8 (NBP2-24917, Novus), NFB (CST8242s, Cell Signaling Technology, USA), p-NFB (CST3033s), SPRY1 (Abcam, ab111523), P-ERK1/2 (AF1018, R&D), ERK1/2 (AF1576, R&D), P-AKT (AF887, R&D), AKT (MAB2055, R&D), P-FAK (MAB4528, R&D), FAK (AF4467, R&D), P-P38 (CST4511), P38 (CST8690), and GAPDH (ab181602) at a ratio of 1:1000 overnight.

Techniques: Protein-Protein interactions, Negative Control, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Western Blot, Over Expression, Plasmid Preparation