pten knockout animals Search Results


pten  (Cell Signaling Technology Inc)
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Cell Signaling Technology Inc pten
Genetic depletion of PRMT6 results in AKT dephosphorylation via impairment of PI3K-AKT signaling. ( A ) BEAS-2B cell lysates were immunoprecipitated with PRMT6 or AKT3 antibody, and the immunoprecipitates were analyzed with AKT3 and PRMT6 immunoblotting as indicated (left two panels). pcDNA3.1D-PRMT6-FLAG and pcDNA3.1D-AKT3-V5 plasmids were co-transfected into BEAS-2B cells. After 48h of transfection, cell lysates were immunoprecipitated with FLAG or V5 antibody, and the immunoprecipitants were analyzed with V5 and FLAG immunoblotting as indicated (right two panels). ( B ) PRMT6 CRISPR/Cas9 KO plasmid and HDR plasmid were applied to establish stable PRMT6 gene knockout BEAS-2B cell line. The knockout efficiency was determined by immunoblotting. The cell lysates of wild type (WT) and PRMT6 stable knockout BEAS-2B cell (PRMT6 -/- ) were applied <t>for</t> <t>pAKT</t> Thr305 , pAKT Ser472 , AKT isoforms 1, 2, 3 and PRMT6 immunoblotting (left panel). Plotted densitometry results of the pAKT Thr305 and pAKT Ser472 in WT and PRMT6 knockout group were presented (right panel). ( C, D ), Cell lysates of WT and PRMT6 -/- BEAS-2B cells were collected and immunoblotted with indicated antibodies. At the right panel of each figure, the plotted densitometry results were presented. ( E ) pcDNA3.1D-His-V5 control plasmid (Vector) and pcDNA3.1D-PRMT6-V5 plasmids were delivered into BEAS-2B cells via electroporation. After 48h, the cell lysates were collected and immunoblotted with indicated antibodies. The plotted densitometry of PI3Kp85, PDK1, <t>PTEN</t> and PRMT6 were presented (middle and right panel). Values represent mean ± SD and “*” denotes p < 0.05. Results were representative of at least n =3 experiments.
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Genetic depletion of PRMT6 results in AKT dephosphorylation via impairment of PI3K-AKT signaling. ( A ) BEAS-2B cell lysates were immunoprecipitated with PRMT6 or AKT3 antibody, and the immunoprecipitates were analyzed with AKT3 and PRMT6 immunoblotting as indicated (left two panels). pcDNA3.1D-PRMT6-FLAG and pcDNA3.1D-AKT3-V5 plasmids were co-transfected into BEAS-2B cells. After 48h of transfection, cell lysates were immunoprecipitated with FLAG or V5 antibody, and the immunoprecipitants were analyzed with V5 and FLAG immunoblotting as indicated (right two panels). ( B ) PRMT6 CRISPR/Cas9 KO plasmid and HDR plasmid were applied to establish stable PRMT6 gene knockout BEAS-2B cell line. The knockout efficiency was determined by immunoblotting. The cell lysates of wild type (WT) and PRMT6 stable knockout BEAS-2B cell (PRMT6 -/- ) were applied for pAKT Thr305 , pAKT Ser472 , AKT isoforms 1, 2, 3 and PRMT6 immunoblotting (left panel). Plotted densitometry results of the pAKT Thr305 and pAKT Ser472 in WT and PRMT6 knockout group were presented (right panel). ( C, D ), Cell lysates of WT and PRMT6 -/- BEAS-2B cells were collected and immunoblotted with indicated antibodies. At the right panel of each figure, the plotted densitometry results were presented. ( E ) pcDNA3.1D-His-V5 control plasmid (Vector) and pcDNA3.1D-PRMT6-V5 plasmids were delivered into BEAS-2B cells via electroporation. After 48h, the cell lysates were collected and immunoblotted with indicated antibodies. The plotted densitometry of PI3Kp85, PDK1, PTEN and PRMT6 were presented (middle and right panel). Values represent mean ± SD and “*” denotes p < 0.05. Results were representative of at least n =3 experiments.

Journal: Aging (Albany NY)

Article Title: Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling

doi: 10.18632/aging.202210

Figure Lengend Snippet: Genetic depletion of PRMT6 results in AKT dephosphorylation via impairment of PI3K-AKT signaling. ( A ) BEAS-2B cell lysates were immunoprecipitated with PRMT6 or AKT3 antibody, and the immunoprecipitates were analyzed with AKT3 and PRMT6 immunoblotting as indicated (left two panels). pcDNA3.1D-PRMT6-FLAG and pcDNA3.1D-AKT3-V5 plasmids were co-transfected into BEAS-2B cells. After 48h of transfection, cell lysates were immunoprecipitated with FLAG or V5 antibody, and the immunoprecipitants were analyzed with V5 and FLAG immunoblotting as indicated (right two panels). ( B ) PRMT6 CRISPR/Cas9 KO plasmid and HDR plasmid were applied to establish stable PRMT6 gene knockout BEAS-2B cell line. The knockout efficiency was determined by immunoblotting. The cell lysates of wild type (WT) and PRMT6 stable knockout BEAS-2B cell (PRMT6 -/- ) were applied for pAKT Thr305 , pAKT Ser472 , AKT isoforms 1, 2, 3 and PRMT6 immunoblotting (left panel). Plotted densitometry results of the pAKT Thr305 and pAKT Ser472 in WT and PRMT6 knockout group were presented (right panel). ( C, D ), Cell lysates of WT and PRMT6 -/- BEAS-2B cells were collected and immunoblotted with indicated antibodies. At the right panel of each figure, the plotted densitometry results were presented. ( E ) pcDNA3.1D-His-V5 control plasmid (Vector) and pcDNA3.1D-PRMT6-V5 plasmids were delivered into BEAS-2B cells via electroporation. After 48h, the cell lysates were collected and immunoblotted with indicated antibodies. The plotted densitometry of PI3Kp85, PDK1, PTEN and PRMT6 were presented (middle and right panel). Values represent mean ± SD and “*” denotes p < 0.05. Results were representative of at least n =3 experiments.

Article Snippet: Antibodies for PRMT6 (Cat#:1461s), AKT1(Cat#: 2938s), AKT2 (Cat#:3063s), AKT3 (Cat#:14982s), mTOR (Cat#:2983s), INPP4b (Cat#: 8450s), PI3Kp85 (Cat#:4257s), and PDK1 (Cat#: 3062s) were from Cell Signaling (Danvers, MA, USA). pAKT Thr305 (Cat#: sc-271966), pAKT Ser472 (Cat#: sc-81433), PTEN (Cat#: sc-79974), and PI3Kp110 (Cat#: sc-8010) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: De-Phosphorylation Assay, Immunoprecipitation, Western Blot, Transfection, CRISPR, Plasmid Preparation, Gene Knockout, Knock-Out, Electroporation

CSE suppresses PI3K/AKT cascade in epithelial cells. ( A , B ) BEAS-2B cells were treated with CSE at various time points as indicated. Cells lysates were analyzed with mTOR, PI3Kp110, PDK1, INPP4b, PI3Kp85, PTEN, PPP2AC, PRMT6, and GAPDH immunoblotting. The densitometry of the blots was presented in the lower panel. ( C , D ) HSAECs cells were treated with CSE at a course of time as indicated. Cells lysates were analyzed with mTOR, PI3Kp110, PDK1, INPP4b, PI3Kp85, PTEN, PPP2AC, PRMT6, and GAPDH immunoblotting. The densitometric results were plotted in the lower panels. ( E , F ) HSAECs cells were exposed to room air or cigarette smoke. Cells lysates were collected and analyzed with indicated antibodies immunoblotting. RA: room air; CS: cigarette smoke. The densitometric blots were showed in the lower panel of each figure. Results were representative of n =3 experiments. Statistical significance was indicated as *: p < .05.

Journal: Aging (Albany NY)

Article Title: Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling

doi: 10.18632/aging.202210

Figure Lengend Snippet: CSE suppresses PI3K/AKT cascade in epithelial cells. ( A , B ) BEAS-2B cells were treated with CSE at various time points as indicated. Cells lysates were analyzed with mTOR, PI3Kp110, PDK1, INPP4b, PI3Kp85, PTEN, PPP2AC, PRMT6, and GAPDH immunoblotting. The densitometry of the blots was presented in the lower panel. ( C , D ) HSAECs cells were treated with CSE at a course of time as indicated. Cells lysates were analyzed with mTOR, PI3Kp110, PDK1, INPP4b, PI3Kp85, PTEN, PPP2AC, PRMT6, and GAPDH immunoblotting. The densitometric results were plotted in the lower panels. ( E , F ) HSAECs cells were exposed to room air or cigarette smoke. Cells lysates were collected and analyzed with indicated antibodies immunoblotting. RA: room air; CS: cigarette smoke. The densitometric blots were showed in the lower panel of each figure. Results were representative of n =3 experiments. Statistical significance was indicated as *: p < .05.

Article Snippet: Antibodies for PRMT6 (Cat#:1461s), AKT1(Cat#: 2938s), AKT2 (Cat#:3063s), AKT3 (Cat#:14982s), mTOR (Cat#:2983s), INPP4b (Cat#: 8450s), PI3Kp85 (Cat#:4257s), and PDK1 (Cat#: 3062s) were from Cell Signaling (Danvers, MA, USA). pAKT Thr305 (Cat#: sc-271966), pAKT Ser472 (Cat#: sc-81433), PTEN (Cat#: sc-79974), and PI3Kp110 (Cat#: sc-8010) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Western Blot

CSE suppresses AKT phosphorylation via PRMT6/PTEN signal transduction. ( A , B ) WT and PRMT6 -/- BEAS-2B cells were treated with CSE at 2, 4, 6h. Western blotting was performed to detect the protein level of PHLPP1, PHLPP2, INPP4b, PI3Kp110, PI3Kp85, PDK1, PTEN, PRMT6 and GAPDH. ( C – E ) The plotted densitometry results of PI3Kp85, PDK1 and PTEN protein expression were presented. ( F – I ) WT and PRMT6 -/- BEAS-2B cells were treated with CSE for 2, 4, 6h. qRT-PCR was performed to detect the mRNA level of PTEN, PDK1, PIK3R1 and PIK3R2 . Results of qRT-PCR were shown as mean ± SD and representative of n =3 experiments. ( J ) Scramble siRNA and three kinds of double strands PTEN-siRNA were transfected into BEAS-2B separately. PTEN expression was detected by western blotting after transfection of PTEN-siRNA for 72hrs. Plotted PTEN protein level was presented in the right panel. ( K , L ) PTEN expression was silenced by DsiRNA in wild type and PRMT6 KO BEAS-2B cells. Western blotting was used to assay the AKT phosphorylation level. Representative blots of PTEN and PRMT6 were shown. ( M ) Cycloheximide (CHX, 100ug/ml) was applied to WT and PRMT6 -/- BEAS-2B cells for 0, 4, 8, 12h. Densitometric results of PTEN and PRMT6 blots were plotted (lower panel). Results were representative of n =3 experiments. Statistics were measured by 1-way and 2-way ANOVA, *: p < 0.05.

Journal: Aging (Albany NY)

Article Title: Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling

doi: 10.18632/aging.202210

Figure Lengend Snippet: CSE suppresses AKT phosphorylation via PRMT6/PTEN signal transduction. ( A , B ) WT and PRMT6 -/- BEAS-2B cells were treated with CSE at 2, 4, 6h. Western blotting was performed to detect the protein level of PHLPP1, PHLPP2, INPP4b, PI3Kp110, PI3Kp85, PDK1, PTEN, PRMT6 and GAPDH. ( C – E ) The plotted densitometry results of PI3Kp85, PDK1 and PTEN protein expression were presented. ( F – I ) WT and PRMT6 -/- BEAS-2B cells were treated with CSE for 2, 4, 6h. qRT-PCR was performed to detect the mRNA level of PTEN, PDK1, PIK3R1 and PIK3R2 . Results of qRT-PCR were shown as mean ± SD and representative of n =3 experiments. ( J ) Scramble siRNA and three kinds of double strands PTEN-siRNA were transfected into BEAS-2B separately. PTEN expression was detected by western blotting after transfection of PTEN-siRNA for 72hrs. Plotted PTEN protein level was presented in the right panel. ( K , L ) PTEN expression was silenced by DsiRNA in wild type and PRMT6 KO BEAS-2B cells. Western blotting was used to assay the AKT phosphorylation level. Representative blots of PTEN and PRMT6 were shown. ( M ) Cycloheximide (CHX, 100ug/ml) was applied to WT and PRMT6 -/- BEAS-2B cells for 0, 4, 8, 12h. Densitometric results of PTEN and PRMT6 blots were plotted (lower panel). Results were representative of n =3 experiments. Statistics were measured by 1-way and 2-way ANOVA, *: p < 0.05.

Article Snippet: Antibodies for PRMT6 (Cat#:1461s), AKT1(Cat#: 2938s), AKT2 (Cat#:3063s), AKT3 (Cat#:14982s), mTOR (Cat#:2983s), INPP4b (Cat#: 8450s), PI3Kp85 (Cat#:4257s), and PDK1 (Cat#: 3062s) were from Cell Signaling (Danvers, MA, USA). pAKT Thr305 (Cat#: sc-271966), pAKT Ser472 (Cat#: sc-81433), PTEN (Cat#: sc-79974), and PI3Kp110 (Cat#: sc-8010) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Transduction, Western Blot, Expressing, Quantitative RT-PCR, Transfection

PRMT6-pAKT axis is crucial in CSE-induced epithelia death. Cigarette smoke reduces PRMT6 at both mRNA and protein levels. Decreased PRMT6 promotes PI3Kp85 expression and increases the protein stability of PTEN. Changes of PI3Kp85 and PTEN, as well as decreased PDK1, reduce the total phosphorylation level of AKT. Lower level of AKT phosphorylation in turn promotes cigarette smoke-induced lung epithelial cell death.

Journal: Aging (Albany NY)

Article Title: Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling

doi: 10.18632/aging.202210

Figure Lengend Snippet: PRMT6-pAKT axis is crucial in CSE-induced epithelia death. Cigarette smoke reduces PRMT6 at both mRNA and protein levels. Decreased PRMT6 promotes PI3Kp85 expression and increases the protein stability of PTEN. Changes of PI3Kp85 and PTEN, as well as decreased PDK1, reduce the total phosphorylation level of AKT. Lower level of AKT phosphorylation in turn promotes cigarette smoke-induced lung epithelial cell death.

Article Snippet: Antibodies for PRMT6 (Cat#:1461s), AKT1(Cat#: 2938s), AKT2 (Cat#:3063s), AKT3 (Cat#:14982s), mTOR (Cat#:2983s), INPP4b (Cat#: 8450s), PI3Kp85 (Cat#:4257s), and PDK1 (Cat#: 3062s) were from Cell Signaling (Danvers, MA, USA). pAKT Thr305 (Cat#: sc-271966), pAKT Ser472 (Cat#: sc-81433), PTEN (Cat#: sc-79974), and PI3Kp110 (Cat#: sc-8010) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing