Journal: eLife

Article Title: Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat

doi: 10.7554/eLife.78044

Figure Lengend Snippet: ( A ) The change in phosphorylation of JNK, ERK, and p38 in S. aureus -infected macrophages. Macrophages were pretreated with DMSO (carrier), Takinib (200 μM), or Amlexanox (100 μM) for 12 hr, then stimulated with S. aureus for 4 hr. Phosphorylation of JNK, ERK, and p38 was observed by Western blot. ( B ) The level of LC3-II was detected in macrophages after S. aureus challenge. Macrophages were pretreated with DMSO, Dynasore (50 μM), SP600125 (200 μM), PD98059 (200 μM), SB203580 (200 μM), or Amlexanox for 12 hr, respectively, then treated with S. aureus (multiplicity of infection [MOI] = 10) for 4 hr. Conversion of LC3-I to LC3-II was monitored by Western blot. GAPDH protein was used as a control. ( C ) The effect activating JNK and ERK1/2 on removal of S. aureus in macrophages. Macrophages were treated with DMSO, SP600125, or PD98059 for 12 hr prior to stimulation with fluorescein isothiocyanate (FITC)-labeled S. aureus (MOI = 10) for 4 hr. The mean fluorescence intensity (MFI) of FITC in macrophages was measured by flow cytometry. ( D ) The expression of TFEB and OPTN was analyzed in macrophages by Western blot. Macrophages were pretreated with DMSO, Dynasore, or Amlexanox for 12 hr, then challenged with S. aureus for 4 hr (MOI = 10). The nucleoprotein was extracted and analyzed by Western blotting to detect the translocation of TFEB. The expression of OPTN was detected by Western blot. LMNB2 and GAPDH were used to normalize samples. Figure 5—source data 1. The original blots of . Figure 5—source data 2. The original blots of . Figure 5—source data 3. The original blots of .

Article Snippet: Antibody , Phospho-p38 MAPK-T180 (Rabbit polyclonal) , ABclonal , Cat#: AP0238 RRID: AB_2771307 , WB (1:1000).

Techniques: Phospho-proteomics, Infection, Western Blot, Control, Labeling, Fluorescence, Flow Cytometry, Expressing, Translocation Assay

Journal: eLife

Article Title: Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat

doi: 10.7554/eLife.78044

Figure Lengend Snippet:

Article Snippet: Antibody , Phospho-p38 MAPK-T180 (Rabbit polyclonal) , ABclonal , Cat#: AP0238 RRID: AB_2771307 , WB (1:1000).

Techniques: Bacteria, Transfection, Construct, Sequencing, Control, Extraction, Software, Staining

Journal: Oncotarget

Article Title: Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways

doi: 10.18632/oncotarget.25481

Figure Lengend Snippet: (A) Cells were pretreated with EGF (100ng/ml) and harvested after the indicated times. Lysates were collected, and p38 MAPK phosphorylation levels were analyzed by Western blotting using the antiphospho-p38 antibody. The same membrane was striped and re-probed with a different antibody for measuring total-p38 MAPK protein levels. (B) Caki-1 cells were preincubated with 400pM of the secreted form of the Klotho (KL) protein at the indicated times followed by EGF (100ng/ml) stimulation for additional 5 min. Cell lysates were subjected to Western blot analysis as described in (A). Plots indicate means ± S.E.M of three independent experiments.

Article Snippet: Rabbit anti-human polyclonal active p38 MAPK antibody and SB203580 were purchased from Promega (Madison, WI).

Techniques: Phospho-proteomics, Western Blot, Membrane

Journal: Oncotarget

Article Title: Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways

doi: 10.18632/oncotarget.25481

Figure Lengend Snippet: (A) Classical in vitro wound healing assay was performed using near-confluent serum-starved Caki-1 cells grown on collagen type 1. Cells were either pretreated with 400pM Klotho (KL) or 1μM p38 MAPK-specific inhibitor SB203580 as positive control, for 60 min followed by EGF (100ng/ml) treatment. Cell culture images shown here were taken at 0 and 24 h. (B) Plots of quantification of the resultant cell motility values were computed by gap surface area measurements for four selected microscopic fields in each assay condition. The degree of migration is expressed as % wound closure compared with zero time point. The results represent means ± S.E.M of three independent experiments. (C) Wound healing assays performed under 3D settings. Images are microphotographs of Caki-1 cells showing hole-closure of “tissue openings” generated with magnetic pattering as described in the materials and method. Cells were pretreated the same way with either Klotho (KL) or SB203580 and stimulated with EGF as described for the classical wound healing assay. (D) Plots of rate closure of holes for Caki-1 cells as a function of EGF exposure in the presence or absence of KL or SB203580.

Article Snippet: Rabbit anti-human polyclonal active p38 MAPK antibody and SB203580 were purchased from Promega (Madison, WI).

Techniques: In Vitro, Wound Healing Assay, Positive Control, Cell Culture, Migration, Generated