rabbit polyclonal antibody against total akt  (Cell Signaling Technology Inc)

Journal: International Journal of Physiology, Pathophysiology and Pharmacology

Article Title: Potential implication of SGK1-dependent activity change in BV-2 microglial cells

doi:

Figure Lengend Snippet: The SGK1 gene is ablated by means of the CRISPR/Cas9 system. (A) The CRISPR/Cas9 system was applied to BV-2 cells, and mutations in SGK1 exon 4 were obtained (see Materials and methods). The wild-type sequence is shown with a target site (underlined) of gRNA. A protospacer adjacent motif (PAM) is shown with white letters in a black box in the wild-type sequence. Expected cleavage site is indicated by arrowhead. Non-identical nucleotides are shown with gray letters. The border between exon 4 and the following intron is shown by a vertical dashed line. Dashes in the sequences represent deleted nucleotides. Those cells underwent immunoblotting using anti-SGK1 (B) and anti-SGK3 (C) antibodies. Protein loading was monitored with actin.

Article Snippet: Reagents and antibodies The following reagents and antibodies were used: lipopolysaccharide (LPS, Escherichia coli 111:B4, Sigma); protease inhibitor cocktail (Sigma); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT, Wako); fluorescein diacetate (FDA, Wako); propidium iodide (PI, Wako); rat monoclonal antibody against CD68 (FA-11) conjugated with fluorescein isothiocyanate (FITC) (BioLegend); rabbit polyclonal antibody against actin (Sigma); rabbit polyclonal antibodies against SGK1, SGK3, phospho-Akt, and total Akt (Cell Signaling), GeneArt Precision gRNA Synthesis Kit (Thermo Fischer Scientific); Cas9 nuclease protein (Thermo Fisher Scientific).

Techniques: CRISPR, Sequencing, Western Blot

Journal: International Journal of Physiology, Pathophysiology and Pharmacology

Article Title: Potential implication of SGK1-dependent activity change in BV-2 microglial cells

doi:

Figure Lengend Snippet: The SGK1 gene is ablated by means of the CRISPR/Cas9 system. (A) The CRISPR/Cas9 system was applied to BV-2 cells, and mutations in SGK1 exon 4 were obtained (see Materials and methods). The wild-type sequence is shown with a target site (underlined) of gRNA. A protospacer adjacent motif (PAM) is shown with white letters in a black box in the wild-type sequence. Expected cleavage site is indicated by arrowhead. Non-identical nucleotides are shown with gray letters. The border between exon 4 and the following intron is shown by a vertical dashed line. Dashes in the sequences represent deleted nucleotides. Those cells underwent immunoblotting using anti-SGK1 (B) and anti-SGK3 (C) antibodies. Protein loading was monitored with actin.

Article Snippet: The following reagents and antibodies were used: lipopolysaccharide (LPS, Escherichia coli 111:B4, Sigma); protease inhibitor cocktail (Sigma); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT, Wako); fluorescein diacetate (FDA, Wako); propidium iodide (PI, Wako); rat monoclonal antibody against CD68 (FA-11) conjugated with fluorescein isothiocyanate (FITC) (BioLegend); rabbit polyclonal antibody against actin (Sigma); rabbit polyclonal antibodies against SGK1, SGK3, phospho-Akt, and total Akt (Cell Signaling), GeneArt Precision gRNA Synthesis Kit (Thermo Fischer Scientific); Cas9 nuclease protein (Thermo Fisher Scientific).

Techniques: CRISPR, Sequencing, Western Blot

Journal: Scientific Reports

Article Title: The effects of artocarpin on wound healing: in vitro and in vivo studies

doi: 10.1038/s41598-017-15876-7

Figure Lengend Snippet: ARTO enhances human fibroblast proliferation and migration through the P38 or JNK signaling pathway. ( a ) Immunohistochemistry was performed to identify vimentin and PCNA (arrows) in wounds on day 15. The cells were pre-treated with MK2006 (MK), PD98059 (PD), SB203580 (SB), or SP600125 (SP) for 1 h and then incubated with ARTO for 24 h. Crystal violet staining ( b ) and a BrdU incorporation assays ( c ) were used to measure GM05386 cell number and proliferation. ( d ) Cell migration was examined via wound healing assays, in which GM05386 cells were wounded by scratch injury (black lines). The wound closure and migration rates were determined. The levels of Akt ( e ), ERK ( f ), P38 ( g ), and JNK ( h ) were determined by western blot analysis. ( i ) There was co-localization between vimentin and phosphorylated P38 or JNK in the wounds on day 15. The data are shown as the means ± S.D. N = 3–6 group and * P < 0.05.

Article Snippet: Polyclonal rabbit IgG antibodies against human GAPDH, β-actin, phospho-/total-P38, phospho-/total-ERK1/2, phospho-/total-JNK, α-actin, collagen, phospho-/total-Akt, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, and anti-rabbit IgG antibodies were purchased from GeneTex (Irvine, CA, USA).

Techniques: Migration, Immunohistochemistry, Incubation, Staining, BrdU Incorporation Assay, Western Blot

Journal: Scientific Reports

Article Title: The effects of artocarpin on wound healing: in vitro and in vivo studies

doi: 10.1038/s41598-017-15876-7

Figure Lengend Snippet: ARTO enhances human keratinocyte proliferation and migration through the ERK or P38 signaling pathway. ( a ) Wounded sections were stained with CK14 and PCNA (arrows). ( b ) Quantitative analysis of PCNA-positive cells. The cells were pre-treated with PD98059 (PD) or SB203580 (SB) for 1 h and then incubated with ARTO for 24 h. Crystal violet staining ( c ), western blot analysis ( d ), and BrdU incorporation assays ( e ) were used to measure keratinocyte proliferation. ( f ) Quantitative analysis of BrdU-positive cells. ( g , h ) The wound closure and migration rates were examined via wound healing assays, in which keratinocytes were wounded by scratch injury (black lines). The levels of Akt ( i ), ERK ( j ), P38 ( k ), and JNK ( l ) were determined by western blot analysis. ( m ) There was co-localization between CK14 and phosphorylated ERK or P38 in the wounds on day 7. The data are shown as the means ± S.D. N = 3–6 wounds/group and * P < 0.05.

Article Snippet: Polyclonal rabbit IgG antibodies against human GAPDH, β-actin, phospho-/total-P38, phospho-/total-ERK1/2, phospho-/total-JNK, α-actin, collagen, phospho-/total-Akt, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, and anti-rabbit IgG antibodies were purchased from GeneTex (Irvine, CA, USA).

Techniques: Migration, Staining, Incubation, Western Blot, BrdU Incorporation Assay

Journal: Scientific Reports

Article Title: The effects of artocarpin on wound healing: in vitro and in vivo studies

doi: 10.1038/s41598-017-15876-7

Figure Lengend Snippet: ARTO enhances angiogenesis through the Akt or P38 signaling pathway. ( a , b ) Wounded sections were stained with CD31, and a quantitative analysis of CD31 level was conducted. ( c ) Western blot analysis of the skin showed that CD31 level was increased in the ARTO-treated wounds on day 15 after wounding. ( d ) Immunohistochemistry was performed to identify CD31 and PCNA (arrows) in wounds on day 15. ( e , f ) MTT, crystal violet staining, and BrdU incorporation assays were used to measure cell viability and proliferation. The levels of Akt ( g ), ERK ( h ), P38 ( i ), and JNK ( j ) were determined by western blot analysis. HUVECs were pre-treated with Akt or MAPK inhibitors for 1 h and then incubated with ARTO for 24 h. ( k ) Crystal violet staining was performed. ( l ) Tube formation and tube length were examined by a Matrigel assay. Confocal image of a tube stained for CD31 (green) and cell nuclei (DAPI, blue). The asterisk indicates the lumen of the tube. ( m ) There was co-localization between CD31 and phosphorylated Akt or P38 in the wounds on day 15. The data are shown as the means ± S.D. N = 3–6 wounds/group and * P < 0.05.

Article Snippet: Polyclonal rabbit IgG antibodies against human GAPDH, β-actin, phospho-/total-P38, phospho-/total-ERK1/2, phospho-/total-JNK, α-actin, collagen, phospho-/total-Akt, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, and anti-rabbit IgG antibodies were purchased from GeneTex (Irvine, CA, USA).

Techniques: Staining, Western Blot, Immunohistochemistry, BrdU Incorporation Assay, Incubation, Matrigel Assay