Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Puerarin attenuates hypoxia-resulted damages in neural stem cells by up-regulating microRNA-214.

doi: 10.1080/21691401.2019.1628040

Figure Lengend Snippet: Figure 6. Puerarin might trigger PI3K/AKT and MEK/ERK signalling pathways by up-regulating miR-214 in hypoxia-treated NSCs. Representative western blotting of total cell lysates immune-blotted and probed with antibodies against (A) p-PI3K, PI3K, p-AKT, AKT, (B) p-MEK, MEK, p-ERK, and ERK. Protein expression was relatively quantified with densitometry with reference to b-actin. NSCs were transfected with miR-214 inhibitor and then pre-treated with puerarin (60 lM) for 24 h before stimulated with hypoxia for 8 h. nsP > .05, P < .05 or P < .01. NC: negative control; miR-214: microRNA-214; qRT-PCR: quantitative reverse transcription PCR; p-PI3K: phosphoTyr458-phosphotylinosital 3 kinase; p-AKT: phosphoSer473-protein kinase B; p-MEK: phosphoSer217/221-mitogen-activated protein kinase kinases; p-ERK: phosphoThr202/Tyr204-extracellular signal regulated protein kinase; NSCs: neural stem cells; SD: standard deviation.

Article Snippet: The membrane was probed with specific antibodies against caspase-3 (Catalog No. C2087-22A; 1:1000) (USBiological, Salem, Massachusetts), caspase-9 (C2088-13B; 1:1000) (USBiological), phosphotylinosital 3 kinase (PI3K) (orb395437; 1:1000) (Biorbyt, Cambridge, UK), phospho (p) Tyr458-PI3K (orb106105, 1:1000) (Biorbyt), protein kinase B (AKT) (3247; 1 lg/mL) (BioVision, Milpitas, CA), pSer473-AKT (3257; 1 lg/mL) (BioVision), mitogen-activated protein kinase (MEK) and pSer217/221-MEK (M2865-02; 1:1000) (USBiological), extracellular signal-regulated protein kinase (ERK) (5AD13MA, 5 mg/mL) (Invitrogen), pThr202/Tyr204-ERK (PA5-37828, 1:1000) (Invitrogen), and b-actin (4970, 1:1000) (Cell Signalling Technology, CST, Beverly, MA) for overnight at 4 C. The primary antibodies were prepared with 5% bull serum albumin (BSA; Millipore) following which, the membrane was incubated with the secondary antibody (7074, 1:5000) (CST) for 1 h at room temperature.

Techniques: Western Blot, Expressing, Transfection, Negative Control, Quantitative RT-PCR, Reverse Transcription, Standard Deviation

Journal: Biochemical and biophysical research communications

Article Title: Phosphoinositide 3-kinase-gamma expression is upregulated in brain microglia and contributes to ischemia-induced microglial activation in acute experimental stroke

doi: 10.1016/j.bbrc.2010.07.116

Figure Lengend Snippet: Cerebral ischemia induces PI3Kγ expression in the brain. A, Reverse transcriptase-PCR analysis of mRNA expression of PI3Kγ subunits (p110γ, p101, p84) in sham-operated and ischemic hemispheres from wild-type (WT) and p110γ knockout (KO) mice 24 h after MCAO. Each lane represents a sample from an individual brain. B, Densitometric analysis of the band shown in A. Data represent mean ± SD from three separate experiments. *P<0.05 versus respective sham controls. C, Western blot analysis of protein expression of the p110γ and p101 in sham-operated and ischemic hemispheres in WT and KO mice. N=3 brain samples per lane. Protein samples extracted from peritoneal leukocytes (PMN) of WT mice used as a positive control. D, Densitometric analysis of the blot shown in C. Data represent mean ± SD from three separate experiments. *P<0.05 versus respective sham controls.

Article Snippet: The p110γ and p101 was detected as previously described [ 20 ], using rabbit polyclonal antibodies against PI3K p110γ (Cell Signaling, 1:1000) and p101 (PIK3R5) (LifeSpan Biosciences).

Techniques: Expressing, Knock-Out, Western Blot, Positive Control

Journal: Biochemical and biophysical research communications

Article Title: Phosphoinositide 3-kinase-gamma expression is upregulated in brain microglia and contributes to ischemia-induced microglial activation in acute experimental stroke

doi: 10.1016/j.bbrc.2010.07.116

Figure Lengend Snippet: Detection of PI3Kγ expression in mouse brain cell lines. A, Reverse transcriptase-PCR analysis of mRNA expression of PI3Kγ subunits (p110γ, p101, p84) in cultured murine brain cell lines. B, Western blot analysis of protein expression of p110γ in cultured murine brain cell lines. All experiments were repeated three times and yielded the same results.

Article Snippet: The p110γ and p101 was detected as previously described [ 20 ], using rabbit polyclonal antibodies against PI3K p110γ (Cell Signaling, 1:1000) and p101 (PIK3R5) (LifeSpan Biosciences).

Techniques: Expressing, Cell Culture, Western Blot