Structured Review

Addgene inc pbabe purol myr ha akt3 plasmid
A, AKT1, 2, 3 and actin as control mRNA expression in A549 and REN cells was evaluated by RT-PCR as reported in in “ ” section. B , AKT1 and 3 protein expression was evaluated by Western blot analysis in REN cells with isoform specific antibodies, tubulin blot confirmed equal loading. Representative of three independent experiments. C , REN cells were transfected with 1 µg/plate of plasmid coding for the constitutive activated form of AKT, <t>Myr-AKT1</t> or 3. 24 hours post transfection REN cells were treated with 23 µM perifosine for 1 hour, then were lysed and equal amounts of proteins were subjected to SDS-PAGE followed by immuno-blotting with specific antibodies to phosphorylated AKT, HA to confirm transfection and tubulin for equal loading. D , The effect of perifosine on cells transfected with Myr- AKT1 or 3 was also assessed on cell proliferation. REN cells were transfected with 1 µg/plate of plasmid coding Myr-AKT1 or 3 and 24 hours post transfection were treated with 23 µM perifosine for 24 hours. At the end of treatment viable cell count was determined. Points , means ± SD of three individual measurements. Perifosine markedly alters AKT membrane translocation, so we next asked whether forced expression of an exogenous Myr-AKT1 or <t>Myr-AKT3</t> could abrogate the effect of perifosine on AKT activating phosphorylations and cell growth. Contradictory data are available in literature on the capability of a constitutively active AKT to overcome perifosine action. While in prostate cancer cells inhibition of AKT phosphorylation was substantially relieved by introduction of a Myr-AKT1, which bypassed the requirement for PH domain-mediated membrane recruitment, in myeloma cells it has been described that perifosine overcome constitutive highly active AKT1 , . We transiently transfected REN cells either with an HA-tagged Myr-AKT1 or Myr-AKT3 and treated them for 24 hours with the IC50 dose of perifosine. Western-blot analysis reported in shows that transfection of both Myr-AKT1 and Myr-AKT3 overcomes perifosine inhibition of AKT phosphorylation in REN cells; only a slight reduction of phosphorylation, probably due to the block of the endogenous proteins, was observed. Cell growth analysis reported in demonstrates that both transfected Myr-AKTs rescued perifosine induced block of cell proliferation.
Pbabe Purol Myr Ha Akt3 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pbabe purol myr ha akt3 plasmid/product/Addgene inc
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pbabe purol myr ha akt3 plasmid - by Bioz Stars, 2024-07
93/100 stars

Images

1) Product Images from "Perifosine as a Potential Novel Anti-Cancer Agent Inhibits EGFR/MET-AKT Axis in Malignant Pleural Mesothelioma"

Article Title: Perifosine as a Potential Novel Anti-Cancer Agent Inhibits EGFR/MET-AKT Axis in Malignant Pleural Mesothelioma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0036856

A, AKT1, 2, 3 and actin as control mRNA expression in A549 and REN cells was evaluated by RT-PCR as reported in in “ ” section. B , AKT1 and 3 protein expression was evaluated by Western blot analysis in REN cells with isoform specific antibodies, tubulin blot confirmed equal loading. Representative of three independent experiments. C , REN cells were transfected with 1 µg/plate of plasmid coding for the constitutive activated form of AKT, Myr-AKT1 or 3. 24 hours post transfection REN cells were treated with 23 µM perifosine for 1 hour, then were lysed and equal amounts of proteins were subjected to SDS-PAGE followed by immuno-blotting with specific antibodies to phosphorylated AKT, HA to confirm transfection and tubulin for equal loading. D , The effect of perifosine on cells transfected with Myr- AKT1 or 3 was also assessed on cell proliferation. REN cells were transfected with 1 µg/plate of plasmid coding Myr-AKT1 or 3 and 24 hours post transfection were treated with 23 µM perifosine for 24 hours. At the end of treatment viable cell count was determined. Points , means ± SD of three individual measurements. Perifosine markedly alters AKT membrane translocation, so we next asked whether forced expression of an exogenous Myr-AKT1 or Myr-AKT3 could abrogate the effect of perifosine on AKT activating phosphorylations and cell growth. Contradictory data are available in literature on the capability of a constitutively active AKT to overcome perifosine action. While in prostate cancer cells inhibition of AKT phosphorylation was substantially relieved by introduction of a Myr-AKT1, which bypassed the requirement for PH domain-mediated membrane recruitment, in myeloma cells it has been described that perifosine overcome constitutive highly active AKT1 , . We transiently transfected REN cells either with an HA-tagged Myr-AKT1 or Myr-AKT3 and treated them for 24 hours with the IC50 dose of perifosine. Western-blot analysis reported in shows that transfection of both Myr-AKT1 and Myr-AKT3 overcomes perifosine inhibition of AKT phosphorylation in REN cells; only a slight reduction of phosphorylation, probably due to the block of the endogenous proteins, was observed. Cell growth analysis reported in demonstrates that both transfected Myr-AKTs rescued perifosine induced block of cell proliferation.
Figure Legend Snippet: A, AKT1, 2, 3 and actin as control mRNA expression in A549 and REN cells was evaluated by RT-PCR as reported in in “ ” section. B , AKT1 and 3 protein expression was evaluated by Western blot analysis in REN cells with isoform specific antibodies, tubulin blot confirmed equal loading. Representative of three independent experiments. C , REN cells were transfected with 1 µg/plate of plasmid coding for the constitutive activated form of AKT, Myr-AKT1 or 3. 24 hours post transfection REN cells were treated with 23 µM perifosine for 1 hour, then were lysed and equal amounts of proteins were subjected to SDS-PAGE followed by immuno-blotting with specific antibodies to phosphorylated AKT, HA to confirm transfection and tubulin for equal loading. D , The effect of perifosine on cells transfected with Myr- AKT1 or 3 was also assessed on cell proliferation. REN cells were transfected with 1 µg/plate of plasmid coding Myr-AKT1 or 3 and 24 hours post transfection were treated with 23 µM perifosine for 24 hours. At the end of treatment viable cell count was determined. Points , means ± SD of three individual measurements. Perifosine markedly alters AKT membrane translocation, so we next asked whether forced expression of an exogenous Myr-AKT1 or Myr-AKT3 could abrogate the effect of perifosine on AKT activating phosphorylations and cell growth. Contradictory data are available in literature on the capability of a constitutively active AKT to overcome perifosine action. While in prostate cancer cells inhibition of AKT phosphorylation was substantially relieved by introduction of a Myr-AKT1, which bypassed the requirement for PH domain-mediated membrane recruitment, in myeloma cells it has been described that perifosine overcome constitutive highly active AKT1 , . We transiently transfected REN cells either with an HA-tagged Myr-AKT1 or Myr-AKT3 and treated them for 24 hours with the IC50 dose of perifosine. Western-blot analysis reported in shows that transfection of both Myr-AKT1 and Myr-AKT3 overcomes perifosine inhibition of AKT phosphorylation in REN cells; only a slight reduction of phosphorylation, probably due to the block of the endogenous proteins, was observed. Cell growth analysis reported in demonstrates that both transfected Myr-AKTs rescued perifosine induced block of cell proliferation.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, SDS Page, Cell Counting, Translocation Assay, Inhibition, Blocking Assay


Structured Review

Addgene inc pbabe puro myr ha akt3
Activated Akt signaling, regardless of isoform type, attenuates expression of EMT-associated transcripts and proteins in MCF10A cells. A. Cells were infected with either control retrovirus Babe (V) or with one expressing Akt1 (1), Akt2 (2) or <t>Akt3</t> (3), respectively. The total RNA was extracted from the respective cells and subjected to RT-qPCR to quantify the transcripts of interest. Heatmap represents relative mRNA levels of EMT-associated transcripts in Akt-infected cells compared to the ones in control counterpart. B. MCF10A cells were lyzed and the resultant mixture was subject to Western immunoblotting using indicated antibodies to assess the levels of proteins of interest. Similar level of GAPDH expression was an indicative of nearly equal amounts of proteins introduced in all samples.
Pbabe Puro Myr Ha Akt3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pbabe puro myr ha akt3/product/Addgene inc
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pbabe puro myr ha akt3 - by Bioz Stars, 2024-07
86/100 stars

Images

1) Product Images from "Dichotomy effects of Akt signaling in breast cancer"

Article Title: Dichotomy effects of Akt signaling in breast cancer

Journal: Molecular Cancer

doi: 10.1186/1476-4598-11-61

Activated Akt signaling, regardless of isoform type, attenuates expression of EMT-associated transcripts and proteins in MCF10A cells. A. Cells were infected with either control retrovirus Babe (V) or with one expressing Akt1 (1), Akt2 (2) or Akt3 (3), respectively. The total RNA was extracted from the respective cells and subjected to RT-qPCR to quantify the transcripts of interest. Heatmap represents relative mRNA levels of EMT-associated transcripts in Akt-infected cells compared to the ones in control counterpart. B. MCF10A cells were lyzed and the resultant mixture was subject to Western immunoblotting using indicated antibodies to assess the levels of proteins of interest. Similar level of GAPDH expression was an indicative of nearly equal amounts of proteins introduced in all samples.
Figure Legend Snippet: Activated Akt signaling, regardless of isoform type, attenuates expression of EMT-associated transcripts and proteins in MCF10A cells. A. Cells were infected with either control retrovirus Babe (V) or with one expressing Akt1 (1), Akt2 (2) or Akt3 (3), respectively. The total RNA was extracted from the respective cells and subjected to RT-qPCR to quantify the transcripts of interest. Heatmap represents relative mRNA levels of EMT-associated transcripts in Akt-infected cells compared to the ones in control counterpart. B. MCF10A cells were lyzed and the resultant mixture was subject to Western immunoblotting using indicated antibodies to assess the levels of proteins of interest. Similar level of GAPDH expression was an indicative of nearly equal amounts of proteins introduced in all samples.

Techniques Used: Expressing, Infection, Quantitative RT-PCR, Western Blot

Activated Akt inhibits TGFβ-induced EMT in an isoform-redundant manner. MCF10A cells were infected with retroviruses to express empty vector (V), Akt1 (1), Akt2 (2) or Akt3 (3) and then cultured in the absence or presence of TGFβ (2 ηg/ml, R & D research) for 3 days followed by RT-qPCR to analyze EMT-associated transcripts. Significant changes in the experimental groups compared to vector control were marked with * or with # (respectively denoting without or with TGFβ treatment) if p < 0.05.
Figure Legend Snippet: Activated Akt inhibits TGFβ-induced EMT in an isoform-redundant manner. MCF10A cells were infected with retroviruses to express empty vector (V), Akt1 (1), Akt2 (2) or Akt3 (3) and then cultured in the absence or presence of TGFβ (2 ηg/ml, R & D research) for 3 days followed by RT-qPCR to analyze EMT-associated transcripts. Significant changes in the experimental groups compared to vector control were marked with * or with # (respectively denoting without or with TGFβ treatment) if p < 0.05.

Techniques Used: Infection, Plasmid Preparation, Cell Culture, Quantitative RT-PCR, IF-P

Akt signaling represses basal motility as well as TGFβ-induced migration in an isoform-independent manner. MCF10A cells expressing vehicle control (V), Akt1 (1), Akt2 (2) or Akt3 (3) were either untreated or stimulated with TGFβ for 3 days. The motility of the resultant cells was assessed either by migration assay at 12-hours ( A and B ) or wound healing assay 20-hours later ( C ).
Figure Legend Snippet: Akt signaling represses basal motility as well as TGFβ-induced migration in an isoform-independent manner. MCF10A cells expressing vehicle control (V), Akt1 (1), Akt2 (2) or Akt3 (3) were either untreated or stimulated with TGFβ for 3 days. The motility of the resultant cells was assessed either by migration assay at 12-hours ( A and B ) or wound healing assay 20-hours later ( C ).

Techniques Used: Migration, Expressing, Wound Healing Assay

Activated Akt signaling decreases breast stem/progenitor cell subpopulations. MCF10A cells were infected with retroviruses to respectively express empty vector (Babe), Akt1, Akt2, or Akt3 and then subject to PE-CD24 and FITC-CD44 double-staining ( A ), ALDEFLUOR assay ( B ), or serially passaged mammosphere formation assay from which mammospheres were counted under a microscope by independent investigators ( C ). * Denotes that the number of mammospheres obtained from Akt-expressing cells differed significantly ( p < 0.05) from the vector controls.
Figure Legend Snippet: Activated Akt signaling decreases breast stem/progenitor cell subpopulations. MCF10A cells were infected with retroviruses to respectively express empty vector (Babe), Akt1, Akt2, or Akt3 and then subject to PE-CD24 and FITC-CD44 double-staining ( A ), ALDEFLUOR assay ( B ), or serially passaged mammosphere formation assay from which mammospheres were counted under a microscope by independent investigators ( C ). * Denotes that the number of mammospheres obtained from Akt-expressing cells differed significantly ( p < 0.05) from the vector controls.

Techniques Used: Infection, Plasmid Preparation, Double Staining, Tube Formation Assay, Microscopy, Expressing

The inhibitory effects of Akt signaling on EMT, cell motility and stem/progenitor cell subfraction can be attenuated in cells that become progressively malignant. A. MI, MII and MIII cells were infected with retroviruses expressing vector control (Babe), Akt1 (1), Akt2 (2), or Akt3 (3), followed by RT-qPCR analysis for assessing expression levels of EMT-associated transcripts. Heatmap represents relative mRNA levels of various transcripts in Akt-expressing cells compared to the ones in control cells set as 1. B. Infected MI, MII, or MIII cells were tested for migration assay 12 hours after seeding on the top chamber of transwell plate and * denotes a significant change ( p < 0.05) as compared to the control cells (Babe). C. Infected MI, MII or MIII cells were subjected to ALDEFLUOR staining (upper) or PE-CD24 plus FITC-CD44 double-staining (lower), followed by FACS analysis to quantify the frequency of ALDH + and CD44 +/high /CD24 -/low subpopulations. * denotes significant (p < 0.05) difference between the Akt-expressing cells and the counterpart control.
Figure Legend Snippet: The inhibitory effects of Akt signaling on EMT, cell motility and stem/progenitor cell subfraction can be attenuated in cells that become progressively malignant. A. MI, MII and MIII cells were infected with retroviruses expressing vector control (Babe), Akt1 (1), Akt2 (2), or Akt3 (3), followed by RT-qPCR analysis for assessing expression levels of EMT-associated transcripts. Heatmap represents relative mRNA levels of various transcripts in Akt-expressing cells compared to the ones in control cells set as 1. B. Infected MI, MII, or MIII cells were tested for migration assay 12 hours after seeding on the top chamber of transwell plate and * denotes a significant change ( p < 0.05) as compared to the control cells (Babe). C. Infected MI, MII or MIII cells were subjected to ALDEFLUOR staining (upper) or PE-CD24 plus FITC-CD44 double-staining (lower), followed by FACS analysis to quantify the frequency of ALDH + and CD44 +/high /CD24 -/low subpopulations. * denotes significant (p < 0.05) difference between the Akt-expressing cells and the counterpart control.

Techniques Used: Infection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Migration, Staining, Double Staining

Activated Akt signaling conveys resistance to chemotherapeutic drugs in an isoform redundant manner. MCF10A cells were infected with retroviruses to respectively express empty vector (Babe), Akt1, Akt2 or Akt3 and then treated with various dosages of paclitaxel ( A ) and doxorubicin ( B ) for 2 and 5 days. % of viable cells was calculated by setting untreated groups as 100% to which the drug treated cells were compared. Akt-overexpression significantly sustained cell viability in response to treatments by cytotoxic agents ( p < 0.05).
Figure Legend Snippet: Activated Akt signaling conveys resistance to chemotherapeutic drugs in an isoform redundant manner. MCF10A cells were infected with retroviruses to respectively express empty vector (Babe), Akt1, Akt2 or Akt3 and then treated with various dosages of paclitaxel ( A ) and doxorubicin ( B ) for 2 and 5 days. % of viable cells was calculated by setting untreated groups as 100% to which the drug treated cells were compared. Akt-overexpression significantly sustained cell viability in response to treatments by cytotoxic agents ( p < 0.05).

Techniques Used: Infection, Plasmid Preparation, Over Expression


Structured Review

Addgene inc pcdna3 myr ha akt3 plasmid
AKT1 Phosphorylates HSF1 on S326 and induces HSF1 activity independent of mTORC1. (A-C) Indicated cell lines were transfected with vector, AKT1, AKT2 or <t>AKT3</t> along with the HSE luciferase reporter for 48 h. Firefly/Renilla ratios were compared across groups. (D) Recombinant proteins were incubated at 30 °C for 2 h in the presence of ATP and subsequently subjected to SDS/PAGE and immunoblotting with indicated antibodies. (E) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cell lysates were subjected to SDS/PAGE and immunoblotting with indicated antibodies. (F-G) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 in the presence or absence of rapamycin (10–20 nM). Cell lysates were subjected to firefly reporter assay (F) or immunoblotting with indicated antibodies (G). Experiments were completed in triplicate and analysed using one-way ANOVA and Tukey’s post-hoc test. Data are presented as mean ± SEM. * P < 0.05.
Pcdna3 Myr Ha Akt3 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcdna3 myr ha akt3 plasmid/product/Addgene inc
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pcdna3 myr ha akt3 plasmid - by Bioz Stars, 2024-07
93/100 stars

Images

1) Product Images from "AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation"

Article Title: AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation

Journal: The FEBS journal

doi: 10.1111/febs.16375

AKT1 Phosphorylates HSF1 on S326 and induces HSF1 activity independent of mTORC1. (A-C) Indicated cell lines were transfected with vector, AKT1, AKT2 or AKT3 along with the HSE luciferase reporter for 48 h. Firefly/Renilla ratios were compared across groups. (D) Recombinant proteins were incubated at 30 °C for 2 h in the presence of ATP and subsequently subjected to SDS/PAGE and immunoblotting with indicated antibodies. (E) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cell lysates were subjected to SDS/PAGE and immunoblotting with indicated antibodies. (F-G) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 in the presence or absence of rapamycin (10–20 nM). Cell lysates were subjected to firefly reporter assay (F) or immunoblotting with indicated antibodies (G). Experiments were completed in triplicate and analysed using one-way ANOVA and Tukey’s post-hoc test. Data are presented as mean ± SEM. * P < 0.05.
Figure Legend Snippet: AKT1 Phosphorylates HSF1 on S326 and induces HSF1 activity independent of mTORC1. (A-C) Indicated cell lines were transfected with vector, AKT1, AKT2 or AKT3 along with the HSE luciferase reporter for 48 h. Firefly/Renilla ratios were compared across groups. (D) Recombinant proteins were incubated at 30 °C for 2 h in the presence of ATP and subsequently subjected to SDS/PAGE and immunoblotting with indicated antibodies. (E) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cell lysates were subjected to SDS/PAGE and immunoblotting with indicated antibodies. (F-G) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 in the presence or absence of rapamycin (10–20 nM). Cell lysates were subjected to firefly reporter assay (F) or immunoblotting with indicated antibodies (G). Experiments were completed in triplicate and analysed using one-way ANOVA and Tukey’s post-hoc test. Data are presented as mean ± SEM. * P < 0.05.

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Recombinant, Incubation, SDS Page, Western Blot, Reporter Assay

AKT1 promotes HSF1 trimerization and DNA binding. (A-B) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cells were fixed with 1 mM EGS for 30 min and lysates were subjected to SDS/PAGE and immunoblotting for HSF1 trimers (A) and AKT isoforms (B). (C) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cells were fixed with formaldehyde, lysed and sonicated prior to ChIP with either control IgG or HSF1 antibodies. ChIP product was subjected to qPCR for indicated gene promoters. (D-E) HEK-293 cells were transfected with either empty vector, AKT1, AKT2 or AKT3 for 48 h. Total RNA was collected from cells and subjected to RT-qPCR for the indicated genes. Experiments were completed in triplicate and analysed using one-way ANOVA and Tukey’s post-hoc test. Data are presented as mean ± SEM. * P < 0.05.
Figure Legend Snippet: AKT1 promotes HSF1 trimerization and DNA binding. (A-B) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cells were fixed with 1 mM EGS for 30 min and lysates were subjected to SDS/PAGE and immunoblotting for HSF1 trimers (A) and AKT isoforms (B). (C) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cells were fixed with formaldehyde, lysed and sonicated prior to ChIP with either control IgG or HSF1 antibodies. ChIP product was subjected to qPCR for indicated gene promoters. (D-E) HEK-293 cells were transfected with either empty vector, AKT1, AKT2 or AKT3 for 48 h. Total RNA was collected from cells and subjected to RT-qPCR for the indicated genes. Experiments were completed in triplicate and analysed using one-way ANOVA and Tukey’s post-hoc test. Data are presented as mean ± SEM. * P < 0.05.

Techniques Used: Binding Assay, Transfection, Plasmid Preparation, SDS Page, Western Blot, Sonication, Quantitative RT-PCR


Structured Review

Addgene inc 1272 pbabepurol myr ha akt3
a Representation of the IL-3 signaling pathway in the murine Ba/F3 cell upon binding of IL-3. b General overview of the established cell lines Ba/F3 <t>myr-Akt1</t> (gray), Ba/F3 myr-Akt2 (red), and Ba/F3 <t>myr-Akt3</t> (yellow). Graph showing the seeded cell number of the Ba/F3 constructs and resulting luminescent signals following treatment with CTG solution compared to the parental system (dotted line, white). The experiments were performed in triplicates with n = 3. Error bars indicate s.d. Immunoblot of myr-Akt isoform systems highlighting the molecular expression levels, activities, and alterations of downstream targets of Akt ( n = 1 biologically independent experiment). c Addressing the Ba/F3 myr-Akt with CAAI borussertib to investigate the sensitivity of the established system. Structure of used inhibitor and schematic representation of the binding mode. Graph showing the remaining viable cells as a percentage with respect to the used concentration of borussertib. Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Immunoblots resolved individual Akt isoforms’ presence and activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). d Addressing the Ba/F3 myr-Akt cells with isoform-selective CAAIs 15a / 16b to investigate selectivity translation into the established system. Graph showing the remaining viable cells as a percentage with respect to the used concentration of 15a and 16b . Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Structure of used inhibitors. Immunoblots resolved isoform selective activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). In c , d the box plot middle line represents the mean value, the box represents the first and third quartile and the error bars indicate s.e. Source Data are provided with this paper.
1272 Pbabepurol Myr Ha Akt3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/1272 pbabepurol myr ha akt3/product/Addgene inc
Average 92 stars, based on 1 article reviews
Price from $9.99 to $1999.99
1272 pbabepurol myr ha akt3 - by Bioz Stars, 2024-07
92/100 stars

Images

1) Product Images from "Cellular model system to dissect the isoform-selectivity of Akt inhibitors"

Article Title: Cellular model system to dissect the isoform-selectivity of Akt inhibitors

Journal: Nature Communications

doi: 10.1038/s41467-021-25512-8

a Representation of the IL-3 signaling pathway in the murine Ba/F3 cell upon binding of IL-3. b General overview of the established cell lines Ba/F3 myr-Akt1 (gray), Ba/F3 myr-Akt2 (red), and Ba/F3 myr-Akt3 (yellow). Graph showing the seeded cell number of the Ba/F3 constructs and resulting luminescent signals following treatment with CTG solution compared to the parental system (dotted line, white). The experiments were performed in triplicates with n = 3. Error bars indicate s.d. Immunoblot of myr-Akt isoform systems highlighting the molecular expression levels, activities, and alterations of downstream targets of Akt ( n = 1 biologically independent experiment). c Addressing the Ba/F3 myr-Akt with CAAI borussertib to investigate the sensitivity of the established system. Structure of used inhibitor and schematic representation of the binding mode. Graph showing the remaining viable cells as a percentage with respect to the used concentration of borussertib. Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Immunoblots resolved individual Akt isoforms’ presence and activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). d Addressing the Ba/F3 myr-Akt cells with isoform-selective CAAIs 15a / 16b to investigate selectivity translation into the established system. Graph showing the remaining viable cells as a percentage with respect to the used concentration of 15a and 16b . Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Structure of used inhibitors. Immunoblots resolved isoform selective activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). In c , d the box plot middle line represents the mean value, the box represents the first and third quartile and the error bars indicate s.e. Source Data are provided with this paper.
Figure Legend Snippet: a Representation of the IL-3 signaling pathway in the murine Ba/F3 cell upon binding of IL-3. b General overview of the established cell lines Ba/F3 myr-Akt1 (gray), Ba/F3 myr-Akt2 (red), and Ba/F3 myr-Akt3 (yellow). Graph showing the seeded cell number of the Ba/F3 constructs and resulting luminescent signals following treatment with CTG solution compared to the parental system (dotted line, white). The experiments were performed in triplicates with n = 3. Error bars indicate s.d. Immunoblot of myr-Akt isoform systems highlighting the molecular expression levels, activities, and alterations of downstream targets of Akt ( n = 1 biologically independent experiment). c Addressing the Ba/F3 myr-Akt with CAAI borussertib to investigate the sensitivity of the established system. Structure of used inhibitor and schematic representation of the binding mode. Graph showing the remaining viable cells as a percentage with respect to the used concentration of borussertib. Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Immunoblots resolved individual Akt isoforms’ presence and activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). d Addressing the Ba/F3 myr-Akt cells with isoform-selective CAAIs 15a / 16b to investigate selectivity translation into the established system. Graph showing the remaining viable cells as a percentage with respect to the used concentration of 15a and 16b . Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Structure of used inhibitors. Immunoblots resolved isoform selective activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). In c , d the box plot middle line represents the mean value, the box represents the first and third quartile and the error bars indicate s.e. Source Data are provided with this paper.

Techniques Used: Binding Assay, Construct, Western Blot, Expressing, Concentration Assay, Activity Assay

Biochemical evaluation of covalent-allosteric Akt inhibitors with the Akt isoforms.
Figure Legend Snippet: Biochemical evaluation of covalent-allosteric Akt inhibitors with the Akt isoforms.

Techniques Used:

a General overview of the closed conformation of protein kinase Akt (kinase domain: gray, PH domain: green; based on PDB: 6HHF) and the druggable binding pockets (ATP-pocket within kinase domain: gray (PDB: 4GV1) , allosteric-interdomain pocket: blue (PDB: 6HHF), PIP 3 -binding pocket within PH domain: gray (PDB 2UVM) ). The variety of the binding site forming residues among the three isoforms are given in percentage; b View of the interdomain binding site in Akt1 (white; PDB 6S9X: with the covalent-allosteric Akt inhibitor 15c (blue)) in comparison to homology models of Akt2 (red) and Akt3 (yellow). The structural changes within the allosteric site and the different side chain residues of each isoform are highlighted; c Modeling studies with recently found pyrazinonic CAAI 16c into Akt isoform homology model highlight structural differences and possible interactions within the allosteric binding site (Akt1(white) PDB: 6S9X; Akt2 (red) and Akt3 (yellow) homology model) . The side view shows an enlarged space in Akt2 and Akt3, ideally to allow the binding of the hydroxyl phenyl moiety (Supplementary Fig. ); d Structure-guided design approach towards a focused set of small molecules for evaluation of Akt isoform-selectivity: trisubstituted, biarylic pyridines function as core scaffold (blue). A benzimidazolone linker (green) is decorated with the Michael acceptor warhead (gray) to address the nucleophilic cysteine residues within the allosteric binding site. Different moieties (red) with various chemical properties and spatial demand should be connected over a defined linkage to the core molecule.
Figure Legend Snippet: a General overview of the closed conformation of protein kinase Akt (kinase domain: gray, PH domain: green; based on PDB: 6HHF) and the druggable binding pockets (ATP-pocket within kinase domain: gray (PDB: 4GV1) , allosteric-interdomain pocket: blue (PDB: 6HHF), PIP 3 -binding pocket within PH domain: gray (PDB 2UVM) ). The variety of the binding site forming residues among the three isoforms are given in percentage; b View of the interdomain binding site in Akt1 (white; PDB 6S9X: with the covalent-allosteric Akt inhibitor 15c (blue)) in comparison to homology models of Akt2 (red) and Akt3 (yellow). The structural changes within the allosteric site and the different side chain residues of each isoform are highlighted; c Modeling studies with recently found pyrazinonic CAAI 16c into Akt isoform homology model highlight structural differences and possible interactions within the allosteric binding site (Akt1(white) PDB: 6S9X; Akt2 (red) and Akt3 (yellow) homology model) . The side view shows an enlarged space in Akt2 and Akt3, ideally to allow the binding of the hydroxyl phenyl moiety (Supplementary Fig. ); d Structure-guided design approach towards a focused set of small molecules for evaluation of Akt isoform-selectivity: trisubstituted, biarylic pyridines function as core scaffold (blue). A benzimidazolone linker (green) is decorated with the Michael acceptor warhead (gray) to address the nucleophilic cysteine residues within the allosteric binding site. Different moieties (red) with various chemical properties and spatial demand should be connected over a defined linkage to the core molecule.

Techniques Used: Binding Assay

Kinetic evaluation of covalent-allosteric Akt ligands with the three Akt isoforms.
Figure Legend Snippet: Kinetic evaluation of covalent-allosteric Akt ligands with the three Akt isoforms.

Techniques Used:


Structured Review

Addgene inc pcdna3 myr ha akt3 plasmid
A-C) Indicated cell lines were transfected with vector, AKT1, AKT2, or <t>AKT3</t> along with the HSE luciferase reporter for 48 hrs. Firefly/Renilla ratios were compared across groups. D) Recombinant proteins were incubated at 30°C for 2 hrs in the presence of ATP and subsequently subjected to SDS-PAGE and immunoblotting with indicated antibodies. E) HEK-293 cells were transfected with vector, AKT1, AKT2, or AKT3 for 48 hrs. Cell lysates were subjected to SDS-PAGE and immunoblotting with indicated antibodies. F-G) HEK-293 cells were transfected with vector, AKT1, AKT2, or AKT3 in the presence or absence of rapamycin (10-20 nM). Cell lysates were subjected to firefly reporter assay (F) or immunblotting with indicated antibodies (G). Experiments were completed in triplicate and analyzed using one-way ANOVA and Tukey’s post-hoc test. Data is presented as mean ± SEM. *P<0.05.
Pcdna3 Myr Ha Akt3 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcdna3 myr ha akt3 plasmid/product/Addgene inc
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pcdna3 myr ha akt3 plasmid - by Bioz Stars, 2024-07
93/100 stars

Images

1) Product Images from "AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation"

Article Title: AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation

Journal: bioRxiv

doi: 10.1101/2020.08.31.275909

A-C) Indicated cell lines were transfected with vector, AKT1, AKT2, or AKT3 along with the HSE luciferase reporter for 48 hrs. Firefly/Renilla ratios were compared across groups. D) Recombinant proteins were incubated at 30°C for 2 hrs in the presence of ATP and subsequently subjected to SDS-PAGE and immunoblotting with indicated antibodies. E) HEK-293 cells were transfected with vector, AKT1, AKT2, or AKT3 for 48 hrs. Cell lysates were subjected to SDS-PAGE and immunoblotting with indicated antibodies. F-G) HEK-293 cells were transfected with vector, AKT1, AKT2, or AKT3 in the presence or absence of rapamycin (10-20 nM). Cell lysates were subjected to firefly reporter assay (F) or immunblotting with indicated antibodies (G). Experiments were completed in triplicate and analyzed using one-way ANOVA and Tukey’s post-hoc test. Data is presented as mean ± SEM. *P<0.05.
Figure Legend Snippet: A-C) Indicated cell lines were transfected with vector, AKT1, AKT2, or AKT3 along with the HSE luciferase reporter for 48 hrs. Firefly/Renilla ratios were compared across groups. D) Recombinant proteins were incubated at 30°C for 2 hrs in the presence of ATP and subsequently subjected to SDS-PAGE and immunoblotting with indicated antibodies. E) HEK-293 cells were transfected with vector, AKT1, AKT2, or AKT3 for 48 hrs. Cell lysates were subjected to SDS-PAGE and immunoblotting with indicated antibodies. F-G) HEK-293 cells were transfected with vector, AKT1, AKT2, or AKT3 in the presence or absence of rapamycin (10-20 nM). Cell lysates were subjected to firefly reporter assay (F) or immunblotting with indicated antibodies (G). Experiments were completed in triplicate and analyzed using one-way ANOVA and Tukey’s post-hoc test. Data is presented as mean ± SEM. *P<0.05.

Techniques Used: Transfection, Plasmid Preparation, Luciferase, Recombinant, Incubation, SDS Page, Western Blot, Reporter Assay

A-B) HEK-293 cells were transfected with vector, AKT1, AKT2, or AKT3 for 48 hrs. Cells were fixed with 1 mM EGS for 30 min and lysates were subjected to SDS-PAGE and immunoblotting for HSF1 trimers (A) and AKT isoforms (B). C) HEK-293 cells were transfected with vector, AKT1, AKT2, or AKT3 for 48 hrs. Cells were fixed with formaldehyde, lysed, and sonicated prior to ChIP with either control IgG or HSF1 antibodies. ChIP product was subjected to qPCR for indicated gene promoters. Experiments were completed in triplicate and analyzed using one-way ANOVA and Tukey’s post-hoc test. Data is presented as mean ± SEM. *P<0.05.
Figure Legend Snippet: A-B) HEK-293 cells were transfected with vector, AKT1, AKT2, or AKT3 for 48 hrs. Cells were fixed with 1 mM EGS for 30 min and lysates were subjected to SDS-PAGE and immunoblotting for HSF1 trimers (A) and AKT isoforms (B). C) HEK-293 cells were transfected with vector, AKT1, AKT2, or AKT3 for 48 hrs. Cells were fixed with formaldehyde, lysed, and sonicated prior to ChIP with either control IgG or HSF1 antibodies. ChIP product was subjected to qPCR for indicated gene promoters. Experiments were completed in triplicate and analyzed using one-way ANOVA and Tukey’s post-hoc test. Data is presented as mean ± SEM. *P<0.05.

Techniques Used: Transfection, Plasmid Preparation, SDS Page, Western Blot, Sonication


Structured Review

Addgene inc myr akt3 pcnda 3 0 ha plasmids
Myr Akt3 Pcnda 3 0 Ha Plasmids, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/myr akt3 pcnda 3 0 ha plasmids/product/Addgene inc
Average 92 stars, based on 1 article reviews
Price from $9.99 to $1999.99
myr akt3 pcnda 3 0 ha plasmids - by Bioz Stars, 2024-07
92/100 stars

Images


Structured Review

Addgene inc 1236 320 pcdna3 myr ha akt3 plasmid 9017
1236 320 Pcdna3 Myr Ha Akt3 Plasmid 9017, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/1236 320 pcdna3 myr ha akt3 plasmid 9017/product/Addgene inc
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
1236 320 pcdna3 myr ha akt3 plasmid 9017 - by Bioz Stars, 2024-07
93/100 stars

Images


Structured Review

Addgene inc 1236 pcdna3 myr ha akt3 plasmid 9017
Akt family members influence GR- and DEX-mediated transactivation of the IEtu1 collapsed promoter. (A) Neuro-2A cells were transfected with the IEtu1 collapsed promoter construct containing the firefly luciferase reporter gene (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1, Akt2, or <t>Akt3</t> (1.0 μg). (B) Neuro-2A cells were transfected with the IEtu1 collapsed promoter (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg). or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped fetal bovine serum (FBS) at approximately 24 h after transfection, and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments, and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.
1236 Pcdna3 Myr Ha Akt3 Plasmid 9017, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/1236 pcdna3 myr ha akt3 plasmid 9017/product/Addgene inc
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
1236 pcdna3 myr ha akt3 plasmid 9017 - by Bioz Stars, 2024-07
93/100 stars

Images

1) Product Images from "Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency"

Article Title: Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency

Journal: Journal of Virology

doi: 10.1128/JVI.00901-20

Akt family members influence GR- and DEX-mediated transactivation of the IEtu1 collapsed promoter. (A) Neuro-2A cells were transfected with the IEtu1 collapsed promoter construct containing the firefly luciferase reporter gene (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1, Akt2, or Akt3 (1.0 μg). (B) Neuro-2A cells were transfected with the IEtu1 collapsed promoter (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg). or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped fetal bovine serum (FBS) at approximately 24 h after transfection, and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments, and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.
Figure Legend Snippet: Akt family members influence GR- and DEX-mediated transactivation of the IEtu1 collapsed promoter. (A) Neuro-2A cells were transfected with the IEtu1 collapsed promoter construct containing the firefly luciferase reporter gene (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1, Akt2, or Akt3 (1.0 μg). (B) Neuro-2A cells were transfected with the IEtu1 collapsed promoter (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg). or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped fetal bovine serum (FBS) at approximately 24 h after transfection, and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments, and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.

Techniques Used: Transfection, Construct, Luciferase, Mutagenesis, Plasmid Preparation, Incubation

Akt1 and Akt2 impair GR-mediated activation of the MMTV-LTR promoter. (A) Neuro-2A cells were transfected with the MMTV-LTR promoter construct (0.5 μg) and, where indicated, plasmids that express GR (1.0 μg), Akt1, Akt2, or Akt3 (1.0 μg). (B) Neuro-2A cells were transfected with the MMTV-LTR promoter construct (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg), or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped FBS at approximately 24 h after transfection and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.
Figure Legend Snippet: Akt1 and Akt2 impair GR-mediated activation of the MMTV-LTR promoter. (A) Neuro-2A cells were transfected with the MMTV-LTR promoter construct (0.5 μg) and, where indicated, plasmids that express GR (1.0 μg), Akt1, Akt2, or Akt3 (1.0 μg). (B) Neuro-2A cells were transfected with the MMTV-LTR promoter construct (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg), or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped FBS at approximately 24 h after transfection and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.

Techniques Used: Activation Assay, Transfection, Construct, Mutagenesis, Plasmid Preparation, Luciferase, Incubation

Akt3 efficiently promotes neurite formation in Neuro-2A cells. Neuro-2A cells were cotransfected with an empty vector (pcDNA3.1) (A), a plasmid expressing Akt1 or Akt2 (Panel B), or Akt3 (C and D) (1 μg plasmid DNA) and a plasmid expressing the lacZ gene (0.1 μg plasmid) to mark transfected cells. (B) A typical result from cells transfected with Akt1 or Akt2. To induce neurite sprouting, 24 h after transfection, cells were seeded into new plates at a low density (2,000 cells/cm2) and then incubated with minimal essential medium (MEM) that contained 0.5% serum for 3 days. Cells were fixed, and β-Gal+ cells were detected by staining.
Figure Legend Snippet: Akt3 efficiently promotes neurite formation in Neuro-2A cells. Neuro-2A cells were cotransfected with an empty vector (pcDNA3.1) (A), a plasmid expressing Akt1 or Akt2 (Panel B), or Akt3 (C and D) (1 μg plasmid DNA) and a plasmid expressing the lacZ gene (0.1 μg plasmid) to mark transfected cells. (B) A typical result from cells transfected with Akt1 or Akt2. To induce neurite sprouting, 24 h after transfection, cells were seeded into new plates at a low density (2,000 cells/cm2) and then incubated with minimal essential medium (MEM) that contained 0.5% serum for 3 days. Cells were fixed, and β-Gal+ cells were detected by staining.

Techniques Used: Plasmid Preparation, Expressing, Transfection, Incubation, Staining

Akt3, but not Akt1or Akt2, significantly increased neurite formation in Neuro-2A cells. The relative efficiency of β-Gal+ cells containing neurites was calculated by dividing the number of β-Gal+ cells with a neurite length at least twice the diameter of the cell by the total number of β-Gal+ cells. The % of β-Gal+ cells with neurites in the control was set at 1. The other samples were compared to the control to obtain the relative efficiency of neurite formation. The average of three independent experiments is shown with the respective standard deviation. An asterisk denotes significant differences (P < 0.05) in β-Gal+ Neuro-2A cells containing neurites following transfection with the Akt family member relative to the number of β-Gal+ Neuro-2A cells with neurites following transfection with an empty vector, as determined by the one-way analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) multiple means comparison tests.
Figure Legend Snippet: Akt3, but not Akt1or Akt2, significantly increased neurite formation in Neuro-2A cells. The relative efficiency of β-Gal+ cells containing neurites was calculated by dividing the number of β-Gal+ cells with a neurite length at least twice the diameter of the cell by the total number of β-Gal+ cells. The % of β-Gal+ cells with neurites in the control was set at 1. The other samples were compared to the control to obtain the relative efficiency of neurite formation. The average of three independent experiments is shown with the respective standard deviation. An asterisk denotes significant differences (P < 0.05) in β-Gal+ Neuro-2A cells containing neurites following transfection with the Akt family member relative to the number of β-Gal+ Neuro-2A cells with neurites following transfection with an empty vector, as determined by the one-way analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) multiple means comparison tests.

Techniques Used: Standard Deviation, Transfection, Plasmid Preparation


Structured Review

Addgene inc 1236 pcdna3 myr ha akt3 plasmid 9017
Akt family members influence GR- and DEX-mediated transactivation of the IEtu1 collapsed promoter. (A) Neuro-2A cells were transfected with the IEtu1 collapsed promoter construct containing the firefly luciferase reporter gene (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1, Akt2, or <t>Akt3</t> (1.0 μg). (B) Neuro-2A cells were transfected with the IEtu1 collapsed promoter (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg). or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped fetal bovine serum (FBS) at approximately 24 h after transfection, and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments, and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.
1236 Pcdna3 Myr Ha Akt3 Plasmid 9017, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/1236 pcdna3 myr ha akt3 plasmid 9017/product/Addgene inc
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
1236 pcdna3 myr ha akt3 plasmid 9017 - by Bioz Stars, 2024-07
86/100 stars

Images

1) Product Images from "Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency"

Article Title: Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency

Journal: Journal of Virology

doi: 10.1128/JVI.00901-20

Akt family members influence GR- and DEX-mediated transactivation of the IEtu1 collapsed promoter. (A) Neuro-2A cells were transfected with the IEtu1 collapsed promoter construct containing the firefly luciferase reporter gene (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1, Akt2, or Akt3 (1.0 μg). (B) Neuro-2A cells were transfected with the IEtu1 collapsed promoter (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg). or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped fetal bovine serum (FBS) at approximately 24 h after transfection, and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments, and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.
Figure Legend Snippet: Akt family members influence GR- and DEX-mediated transactivation of the IEtu1 collapsed promoter. (A) Neuro-2A cells were transfected with the IEtu1 collapsed promoter construct containing the firefly luciferase reporter gene (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1, Akt2, or Akt3 (1.0 μg). (B) Neuro-2A cells were transfected with the IEtu1 collapsed promoter (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg). or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped fetal bovine serum (FBS) at approximately 24 h after transfection, and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments, and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.

Techniques Used: Transfection, Construct, Luciferase, Mutagenesis, Plasmid Preparation, Incubation

Akt1 and Akt2 impair GR-mediated activation of the MMTV-LTR promoter. (A) Neuro-2A cells were transfected with the MMTV-LTR promoter construct (0.5 μg) and, where indicated, plasmids that express GR (1.0 μg), Akt1, Akt2, or Akt3 (1.0 μg). (B) Neuro-2A cells were transfected with the MMTV-LTR promoter construct (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg), or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped FBS at approximately 24 h after transfection and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.
Figure Legend Snippet: Akt1 and Akt2 impair GR-mediated activation of the MMTV-LTR promoter. (A) Neuro-2A cells were transfected with the MMTV-LTR promoter construct (0.5 μg) and, where indicated, plasmids that express GR (1.0 μg), Akt1, Akt2, or Akt3 (1.0 μg). (B) Neuro-2A cells were transfected with the MMTV-LTR promoter construct (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg), or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped FBS at approximately 24 h after transfection and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.

Techniques Used: Activation Assay, Transfection, Construct, Mutagenesis, Plasmid Preparation, Luciferase, Incubation

Akt3 efficiently promotes neurite formation in Neuro-2A cells. Neuro-2A cells were cotransfected with an empty vector (pcDNA3.1) (A), a plasmid expressing Akt1 or Akt2 (Panel B), or Akt3 (C and D) (1 μg plasmid DNA) and a plasmid expressing the lacZ gene (0.1 μg plasmid) to mark transfected cells. (B) A typical result from cells transfected with Akt1 or Akt2. To induce neurite sprouting, 24 h after transfection, cells were seeded into new plates at a low density (2,000 cells/cm2) and then incubated with minimal essential medium (MEM) that contained 0.5% serum for 3 days. Cells were fixed, and β-Gal+ cells were detected by staining.
Figure Legend Snippet: Akt3 efficiently promotes neurite formation in Neuro-2A cells. Neuro-2A cells were cotransfected with an empty vector (pcDNA3.1) (A), a plasmid expressing Akt1 or Akt2 (Panel B), or Akt3 (C and D) (1 μg plasmid DNA) and a plasmid expressing the lacZ gene (0.1 μg plasmid) to mark transfected cells. (B) A typical result from cells transfected with Akt1 or Akt2. To induce neurite sprouting, 24 h after transfection, cells were seeded into new plates at a low density (2,000 cells/cm2) and then incubated with minimal essential medium (MEM) that contained 0.5% serum for 3 days. Cells were fixed, and β-Gal+ cells were detected by staining.

Techniques Used: Plasmid Preparation, Expressing, Transfection, Incubation, Staining

Akt3, but not Akt1or Akt2, significantly increased neurite formation in Neuro-2A cells. The relative efficiency of β-Gal+ cells containing neurites was calculated by dividing the number of β-Gal+ cells with a neurite length at least twice the diameter of the cell by the total number of β-Gal+ cells. The % of β-Gal+ cells with neurites in the control was set at 1. The other samples were compared to the control to obtain the relative efficiency of neurite formation. The average of three independent experiments is shown with the respective standard deviation. An asterisk denotes significant differences (P < 0.05) in β-Gal+ Neuro-2A cells containing neurites following transfection with the Akt family member relative to the number of β-Gal+ Neuro-2A cells with neurites following transfection with an empty vector, as determined by the one-way analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) multiple means comparison tests.
Figure Legend Snippet: Akt3, but not Akt1or Akt2, significantly increased neurite formation in Neuro-2A cells. The relative efficiency of β-Gal+ cells containing neurites was calculated by dividing the number of β-Gal+ cells with a neurite length at least twice the diameter of the cell by the total number of β-Gal+ cells. The % of β-Gal+ cells with neurites in the control was set at 1. The other samples were compared to the control to obtain the relative efficiency of neurite formation. The average of three independent experiments is shown with the respective standard deviation. An asterisk denotes significant differences (P < 0.05) in β-Gal+ Neuro-2A cells containing neurites following transfection with the Akt family member relative to the number of β-Gal+ Neuro-2A cells with neurites following transfection with an empty vector, as determined by the one-way analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) multiple means comparison tests.

Techniques Used: Standard Deviation, Transfection, Plasmid Preparation


Structured Review

Addgene inc 1236 pcdna3 myr ha akt3
1236 Pcdna3 Myr Ha Akt3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/1236 pcdna3 myr ha akt3/product/Addgene inc
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
1236 pcdna3 myr ha akt3 - by Bioz Stars, 2024-07
93/100 stars

Images

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Addgene inc pbabe purol myr ha akt3 plasmid
    A, AKT1, 2, 3 and actin as control mRNA expression in A549 and REN cells was evaluated by RT-PCR as reported in in “ ” section. B , AKT1 and 3 protein expression was evaluated by Western blot analysis in REN cells with isoform specific antibodies, tubulin blot confirmed equal loading. Representative of three independent experiments. C , REN cells were transfected with 1 µg/plate of plasmid coding for the constitutive activated form of AKT, <t>Myr-AKT1</t> or 3. 24 hours post transfection REN cells were treated with 23 µM perifosine for 1 hour, then were lysed and equal amounts of proteins were subjected to SDS-PAGE followed by immuno-blotting with specific antibodies to phosphorylated AKT, HA to confirm transfection and tubulin for equal loading. D , The effect of perifosine on cells transfected with Myr- AKT1 or 3 was also assessed on cell proliferation. REN cells were transfected with 1 µg/plate of plasmid coding Myr-AKT1 or 3 and 24 hours post transfection were treated with 23 µM perifosine for 24 hours. At the end of treatment viable cell count was determined. Points , means ± SD of three individual measurements. Perifosine markedly alters AKT membrane translocation, so we next asked whether forced expression of an exogenous Myr-AKT1 or <t>Myr-AKT3</t> could abrogate the effect of perifosine on AKT activating phosphorylations and cell growth. Contradictory data are available in literature on the capability of a constitutively active AKT to overcome perifosine action. While in prostate cancer cells inhibition of AKT phosphorylation was substantially relieved by introduction of a Myr-AKT1, which bypassed the requirement for PH domain-mediated membrane recruitment, in myeloma cells it has been described that perifosine overcome constitutive highly active AKT1 , . We transiently transfected REN cells either with an HA-tagged Myr-AKT1 or Myr-AKT3 and treated them for 24 hours with the IC50 dose of perifosine. Western-blot analysis reported in shows that transfection of both Myr-AKT1 and Myr-AKT3 overcomes perifosine inhibition of AKT phosphorylation in REN cells; only a slight reduction of phosphorylation, probably due to the block of the endogenous proteins, was observed. Cell growth analysis reported in demonstrates that both transfected Myr-AKTs rescued perifosine induced block of cell proliferation.
    Pbabe Purol Myr Ha Akt3 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbabe purol myr ha akt3 plasmid/product/Addgene inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbabe purol myr ha akt3 plasmid - by Bioz Stars, 2024-07
    93/100 stars
      Buy from Supplier

    86
    Addgene inc pbabe puro myr ha akt3
    Activated Akt signaling, regardless of isoform type, attenuates expression of EMT-associated transcripts and proteins in MCF10A cells. A. Cells were infected with either control retrovirus Babe (V) or with one expressing Akt1 (1), Akt2 (2) or <t>Akt3</t> (3), respectively. The total RNA was extracted from the respective cells and subjected to RT-qPCR to quantify the transcripts of interest. Heatmap represents relative mRNA levels of EMT-associated transcripts in Akt-infected cells compared to the ones in control counterpart. B. MCF10A cells were lyzed and the resultant mixture was subject to Western immunoblotting using indicated antibodies to assess the levels of proteins of interest. Similar level of GAPDH expression was an indicative of nearly equal amounts of proteins introduced in all samples.
    Pbabe Puro Myr Ha Akt3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbabe puro myr ha akt3/product/Addgene inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbabe puro myr ha akt3 - by Bioz Stars, 2024-07
    86/100 stars
      Buy from Supplier

    93
    Addgene inc pcdna3 myr ha akt3 plasmid
    AKT1 Phosphorylates HSF1 on S326 and induces HSF1 activity independent of mTORC1. (A-C) Indicated cell lines were transfected with vector, AKT1, AKT2 or <t>AKT3</t> along with the HSE luciferase reporter for 48 h. Firefly/Renilla ratios were compared across groups. (D) Recombinant proteins were incubated at 30 °C for 2 h in the presence of ATP and subsequently subjected to SDS/PAGE and immunoblotting with indicated antibodies. (E) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cell lysates were subjected to SDS/PAGE and immunoblotting with indicated antibodies. (F-G) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 in the presence or absence of rapamycin (10–20 nM). Cell lysates were subjected to firefly reporter assay (F) or immunoblotting with indicated antibodies (G). Experiments were completed in triplicate and analysed using one-way ANOVA and Tukey’s post-hoc test. Data are presented as mean ± SEM. * P < 0.05.
    Pcdna3 Myr Ha Akt3 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 myr ha akt3 plasmid/product/Addgene inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcdna3 myr ha akt3 plasmid - by Bioz Stars, 2024-07
    93/100 stars
      Buy from Supplier

    92
    Addgene inc 1272 pbabepurol myr ha akt3
    a Representation of the IL-3 signaling pathway in the murine Ba/F3 cell upon binding of IL-3. b General overview of the established cell lines Ba/F3 <t>myr-Akt1</t> (gray), Ba/F3 myr-Akt2 (red), and Ba/F3 <t>myr-Akt3</t> (yellow). Graph showing the seeded cell number of the Ba/F3 constructs and resulting luminescent signals following treatment with CTG solution compared to the parental system (dotted line, white). The experiments were performed in triplicates with n = 3. Error bars indicate s.d. Immunoblot of myr-Akt isoform systems highlighting the molecular expression levels, activities, and alterations of downstream targets of Akt ( n = 1 biologically independent experiment). c Addressing the Ba/F3 myr-Akt with CAAI borussertib to investigate the sensitivity of the established system. Structure of used inhibitor and schematic representation of the binding mode. Graph showing the remaining viable cells as a percentage with respect to the used concentration of borussertib. Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Immunoblots resolved individual Akt isoforms’ presence and activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). d Addressing the Ba/F3 myr-Akt cells with isoform-selective CAAIs 15a / 16b to investigate selectivity translation into the established system. Graph showing the remaining viable cells as a percentage with respect to the used concentration of 15a and 16b . Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Structure of used inhibitors. Immunoblots resolved isoform selective activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). In c , d the box plot middle line represents the mean value, the box represents the first and third quartile and the error bars indicate s.e. Source Data are provided with this paper.
    1272 Pbabepurol Myr Ha Akt3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/1272 pbabepurol myr ha akt3/product/Addgene inc
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    1272 pbabepurol myr ha akt3 - by Bioz Stars, 2024-07
    92/100 stars
      Buy from Supplier

    92
    Addgene inc myr akt3 pcnda 3 0 ha plasmids
    a Representation of the IL-3 signaling pathway in the murine Ba/F3 cell upon binding of IL-3. b General overview of the established cell lines Ba/F3 <t>myr-Akt1</t> (gray), Ba/F3 myr-Akt2 (red), and Ba/F3 <t>myr-Akt3</t> (yellow). Graph showing the seeded cell number of the Ba/F3 constructs and resulting luminescent signals following treatment with CTG solution compared to the parental system (dotted line, white). The experiments were performed in triplicates with n = 3. Error bars indicate s.d. Immunoblot of myr-Akt isoform systems highlighting the molecular expression levels, activities, and alterations of downstream targets of Akt ( n = 1 biologically independent experiment). c Addressing the Ba/F3 myr-Akt with CAAI borussertib to investigate the sensitivity of the established system. Structure of used inhibitor and schematic representation of the binding mode. Graph showing the remaining viable cells as a percentage with respect to the used concentration of borussertib. Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Immunoblots resolved individual Akt isoforms’ presence and activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). d Addressing the Ba/F3 myr-Akt cells with isoform-selective CAAIs 15a / 16b to investigate selectivity translation into the established system. Graph showing the remaining viable cells as a percentage with respect to the used concentration of 15a and 16b . Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Structure of used inhibitors. Immunoblots resolved isoform selective activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). In c , d the box plot middle line represents the mean value, the box represents the first and third quartile and the error bars indicate s.e. Source Data are provided with this paper.
    Myr Akt3 Pcnda 3 0 Ha Plasmids, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/myr akt3 pcnda 3 0 ha plasmids/product/Addgene inc
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    myr akt3 pcnda 3 0 ha plasmids - by Bioz Stars, 2024-07
    92/100 stars
      Buy from Supplier

    93
    Addgene inc 1236 320 pcdna3 myr ha akt3 plasmid 9017
    a Representation of the IL-3 signaling pathway in the murine Ba/F3 cell upon binding of IL-3. b General overview of the established cell lines Ba/F3 <t>myr-Akt1</t> (gray), Ba/F3 myr-Akt2 (red), and Ba/F3 <t>myr-Akt3</t> (yellow). Graph showing the seeded cell number of the Ba/F3 constructs and resulting luminescent signals following treatment with CTG solution compared to the parental system (dotted line, white). The experiments were performed in triplicates with n = 3. Error bars indicate s.d. Immunoblot of myr-Akt isoform systems highlighting the molecular expression levels, activities, and alterations of downstream targets of Akt ( n = 1 biologically independent experiment). c Addressing the Ba/F3 myr-Akt with CAAI borussertib to investigate the sensitivity of the established system. Structure of used inhibitor and schematic representation of the binding mode. Graph showing the remaining viable cells as a percentage with respect to the used concentration of borussertib. Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Immunoblots resolved individual Akt isoforms’ presence and activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). d Addressing the Ba/F3 myr-Akt cells with isoform-selective CAAIs 15a / 16b to investigate selectivity translation into the established system. Graph showing the remaining viable cells as a percentage with respect to the used concentration of 15a and 16b . Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Structure of used inhibitors. Immunoblots resolved isoform selective activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). In c , d the box plot middle line represents the mean value, the box represents the first and third quartile and the error bars indicate s.e. Source Data are provided with this paper.
    1236 320 Pcdna3 Myr Ha Akt3 Plasmid 9017, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/1236 320 pcdna3 myr ha akt3 plasmid 9017/product/Addgene inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    1236 320 pcdna3 myr ha akt3 plasmid 9017 - by Bioz Stars, 2024-07
    93/100 stars
      Buy from Supplier

    93
    Addgene inc 1236 pcdna3 myr ha akt3 plasmid 9017
    Akt family members influence GR- and DEX-mediated transactivation of the IEtu1 collapsed promoter. (A) Neuro-2A cells were transfected with the IEtu1 collapsed promoter construct containing the firefly luciferase reporter gene (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1, Akt2, or <t>Akt3</t> (1.0 μg). (B) Neuro-2A cells were transfected with the IEtu1 collapsed promoter (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg). or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped fetal bovine serum (FBS) at approximately 24 h after transfection, and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments, and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.
    1236 Pcdna3 Myr Ha Akt3 Plasmid 9017, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/1236 pcdna3 myr ha akt3 plasmid 9017/product/Addgene inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    1236 pcdna3 myr ha akt3 plasmid 9017 - by Bioz Stars, 2024-07
    93/100 stars
      Buy from Supplier

    93
    Addgene inc 1236 pcdna3 myr ha akt3
    Akt family members influence GR- and DEX-mediated transactivation of the IEtu1 collapsed promoter. (A) Neuro-2A cells were transfected with the IEtu1 collapsed promoter construct containing the firefly luciferase reporter gene (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1, Akt2, or <t>Akt3</t> (1.0 μg). (B) Neuro-2A cells were transfected with the IEtu1 collapsed promoter (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg). or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped fetal bovine serum (FBS) at approximately 24 h after transfection, and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments, and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.
    1236 Pcdna3 Myr Ha Akt3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/1236 pcdna3 myr ha akt3/product/Addgene inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    1236 pcdna3 myr ha akt3 - by Bioz Stars, 2024-07
    93/100 stars
      Buy from Supplier

    Image Search Results


    A, AKT1, 2, 3 and actin as control mRNA expression in A549 and REN cells was evaluated by RT-PCR as reported in in “ ” section. B , AKT1 and 3 protein expression was evaluated by Western blot analysis in REN cells with isoform specific antibodies, tubulin blot confirmed equal loading. Representative of three independent experiments. C , REN cells were transfected with 1 µg/plate of plasmid coding for the constitutive activated form of AKT, Myr-AKT1 or 3. 24 hours post transfection REN cells were treated with 23 µM perifosine for 1 hour, then were lysed and equal amounts of proteins were subjected to SDS-PAGE followed by immuno-blotting with specific antibodies to phosphorylated AKT, HA to confirm transfection and tubulin for equal loading. D , The effect of perifosine on cells transfected with Myr- AKT1 or 3 was also assessed on cell proliferation. REN cells were transfected with 1 µg/plate of plasmid coding Myr-AKT1 or 3 and 24 hours post transfection were treated with 23 µM perifosine for 24 hours. At the end of treatment viable cell count was determined. Points , means ± SD of three individual measurements. Perifosine markedly alters AKT membrane translocation, so we next asked whether forced expression of an exogenous Myr-AKT1 or Myr-AKT3 could abrogate the effect of perifosine on AKT activating phosphorylations and cell growth. Contradictory data are available in literature on the capability of a constitutively active AKT to overcome perifosine action. While in prostate cancer cells inhibition of AKT phosphorylation was substantially relieved by introduction of a Myr-AKT1, which bypassed the requirement for PH domain-mediated membrane recruitment, in myeloma cells it has been described that perifosine overcome constitutive highly active AKT1 , . We transiently transfected REN cells either with an HA-tagged Myr-AKT1 or Myr-AKT3 and treated them for 24 hours with the IC50 dose of perifosine. Western-blot analysis reported in shows that transfection of both Myr-AKT1 and Myr-AKT3 overcomes perifosine inhibition of AKT phosphorylation in REN cells; only a slight reduction of phosphorylation, probably due to the block of the endogenous proteins, was observed. Cell growth analysis reported in demonstrates that both transfected Myr-AKTs rescued perifosine induced block of cell proliferation.

    Journal: PLoS ONE

    Article Title: Perifosine as a Potential Novel Anti-Cancer Agent Inhibits EGFR/MET-AKT Axis in Malignant Pleural Mesothelioma

    doi: 10.1371/journal.pone.0036856

    Figure Lengend Snippet: A, AKT1, 2, 3 and actin as control mRNA expression in A549 and REN cells was evaluated by RT-PCR as reported in in “ ” section. B , AKT1 and 3 protein expression was evaluated by Western blot analysis in REN cells with isoform specific antibodies, tubulin blot confirmed equal loading. Representative of three independent experiments. C , REN cells were transfected with 1 µg/plate of plasmid coding for the constitutive activated form of AKT, Myr-AKT1 or 3. 24 hours post transfection REN cells were treated with 23 µM perifosine for 1 hour, then were lysed and equal amounts of proteins were subjected to SDS-PAGE followed by immuno-blotting with specific antibodies to phosphorylated AKT, HA to confirm transfection and tubulin for equal loading. D , The effect of perifosine on cells transfected with Myr- AKT1 or 3 was also assessed on cell proliferation. REN cells were transfected with 1 µg/plate of plasmid coding Myr-AKT1 or 3 and 24 hours post transfection were treated with 23 µM perifosine for 24 hours. At the end of treatment viable cell count was determined. Points , means ± SD of three individual measurements. Perifosine markedly alters AKT membrane translocation, so we next asked whether forced expression of an exogenous Myr-AKT1 or Myr-AKT3 could abrogate the effect of perifosine on AKT activating phosphorylations and cell growth. Contradictory data are available in literature on the capability of a constitutively active AKT to overcome perifosine action. While in prostate cancer cells inhibition of AKT phosphorylation was substantially relieved by introduction of a Myr-AKT1, which bypassed the requirement for PH domain-mediated membrane recruitment, in myeloma cells it has been described that perifosine overcome constitutive highly active AKT1 , . We transiently transfected REN cells either with an HA-tagged Myr-AKT1 or Myr-AKT3 and treated them for 24 hours with the IC50 dose of perifosine. Western-blot analysis reported in shows that transfection of both Myr-AKT1 and Myr-AKT3 overcomes perifosine inhibition of AKT phosphorylation in REN cells; only a slight reduction of phosphorylation, probably due to the block of the endogenous proteins, was observed. Cell growth analysis reported in demonstrates that both transfected Myr-AKTs rescued perifosine induced block of cell proliferation.

    Article Snippet: Cells grown to 80% confluence in tissue culture dishes were transiently transfected with pcDNA3 Myr-HA-AKT1 or pBABE puroL Myr-HA-AKT3 plasmid encoding for myristilated constitutively active form of AKTs from Addgene by the LipofectAMINE reagent as described by the manufacturer.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, SDS Page, Cell Counting, Translocation Assay, Inhibition, Blocking Assay

    Activated Akt signaling, regardless of isoform type, attenuates expression of EMT-associated transcripts and proteins in MCF10A cells. A. Cells were infected with either control retrovirus Babe (V) or with one expressing Akt1 (1), Akt2 (2) or Akt3 (3), respectively. The total RNA was extracted from the respective cells and subjected to RT-qPCR to quantify the transcripts of interest. Heatmap represents relative mRNA levels of EMT-associated transcripts in Akt-infected cells compared to the ones in control counterpart. B. MCF10A cells were lyzed and the resultant mixture was subject to Western immunoblotting using indicated antibodies to assess the levels of proteins of interest. Similar level of GAPDH expression was an indicative of nearly equal amounts of proteins introduced in all samples.

    Journal: Molecular Cancer

    Article Title: Dichotomy effects of Akt signaling in breast cancer

    doi: 10.1186/1476-4598-11-61

    Figure Lengend Snippet: Activated Akt signaling, regardless of isoform type, attenuates expression of EMT-associated transcripts and proteins in MCF10A cells. A. Cells were infected with either control retrovirus Babe (V) or with one expressing Akt1 (1), Akt2 (2) or Akt3 (3), respectively. The total RNA was extracted from the respective cells and subjected to RT-qPCR to quantify the transcripts of interest. Heatmap represents relative mRNA levels of EMT-associated transcripts in Akt-infected cells compared to the ones in control counterpart. B. MCF10A cells were lyzed and the resultant mixture was subject to Western immunoblotting using indicated antibodies to assess the levels of proteins of interest. Similar level of GAPDH expression was an indicative of nearly equal amounts of proteins introduced in all samples.

    Article Snippet: Antibiotic-antimycotic (100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphoterincin) was routinely included in medium to prevent microbial contamination. pBabe-Puro, pBabe-Puro-Myr-Flag-Akt1 [ ], pBabe-Puro-Myr-HA-Akt2, pBabe-Puro-Myr-HA-Akt3, pBabe-Bleo, and pBabe-Bleo-IGF-1R, were purchased from Addgene Inc. To obtain infectious retrovirions prior to transducing Myr-Akt into target cells, retroviral vectors were first introduced into packaging cells known as Phoenix™ Ampho (Orbigen, Inc.) by a calcium phosphate transfection method.

    Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot

    Activated Akt inhibits TGFβ-induced EMT in an isoform-redundant manner. MCF10A cells were infected with retroviruses to express empty vector (V), Akt1 (1), Akt2 (2) or Akt3 (3) and then cultured in the absence or presence of TGFβ (2 ηg/ml, R & D research) for 3 days followed by RT-qPCR to analyze EMT-associated transcripts. Significant changes in the experimental groups compared to vector control were marked with * or with # (respectively denoting without or with TGFβ treatment) if p < 0.05.

    Journal: Molecular Cancer

    Article Title: Dichotomy effects of Akt signaling in breast cancer

    doi: 10.1186/1476-4598-11-61

    Figure Lengend Snippet: Activated Akt inhibits TGFβ-induced EMT in an isoform-redundant manner. MCF10A cells were infected with retroviruses to express empty vector (V), Akt1 (1), Akt2 (2) or Akt3 (3) and then cultured in the absence or presence of TGFβ (2 ηg/ml, R & D research) for 3 days followed by RT-qPCR to analyze EMT-associated transcripts. Significant changes in the experimental groups compared to vector control were marked with * or with # (respectively denoting without or with TGFβ treatment) if p < 0.05.

    Article Snippet: Antibiotic-antimycotic (100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphoterincin) was routinely included in medium to prevent microbial contamination. pBabe-Puro, pBabe-Puro-Myr-Flag-Akt1 [ ], pBabe-Puro-Myr-HA-Akt2, pBabe-Puro-Myr-HA-Akt3, pBabe-Bleo, and pBabe-Bleo-IGF-1R, were purchased from Addgene Inc. To obtain infectious retrovirions prior to transducing Myr-Akt into target cells, retroviral vectors were first introduced into packaging cells known as Phoenix™ Ampho (Orbigen, Inc.) by a calcium phosphate transfection method.

    Techniques: Infection, Plasmid Preparation, Cell Culture, Quantitative RT-PCR, IF-P

    Akt signaling represses basal motility as well as TGFβ-induced migration in an isoform-independent manner. MCF10A cells expressing vehicle control (V), Akt1 (1), Akt2 (2) or Akt3 (3) were either untreated or stimulated with TGFβ for 3 days. The motility of the resultant cells was assessed either by migration assay at 12-hours ( A and B ) or wound healing assay 20-hours later ( C ).

    Journal: Molecular Cancer

    Article Title: Dichotomy effects of Akt signaling in breast cancer

    doi: 10.1186/1476-4598-11-61

    Figure Lengend Snippet: Akt signaling represses basal motility as well as TGFβ-induced migration in an isoform-independent manner. MCF10A cells expressing vehicle control (V), Akt1 (1), Akt2 (2) or Akt3 (3) were either untreated or stimulated with TGFβ for 3 days. The motility of the resultant cells was assessed either by migration assay at 12-hours ( A and B ) or wound healing assay 20-hours later ( C ).

    Article Snippet: Antibiotic-antimycotic (100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphoterincin) was routinely included in medium to prevent microbial contamination. pBabe-Puro, pBabe-Puro-Myr-Flag-Akt1 [ ], pBabe-Puro-Myr-HA-Akt2, pBabe-Puro-Myr-HA-Akt3, pBabe-Bleo, and pBabe-Bleo-IGF-1R, were purchased from Addgene Inc. To obtain infectious retrovirions prior to transducing Myr-Akt into target cells, retroviral vectors were first introduced into packaging cells known as Phoenix™ Ampho (Orbigen, Inc.) by a calcium phosphate transfection method.

    Techniques: Migration, Expressing, Wound Healing Assay

    Activated Akt signaling decreases breast stem/progenitor cell subpopulations. MCF10A cells were infected with retroviruses to respectively express empty vector (Babe), Akt1, Akt2, or Akt3 and then subject to PE-CD24 and FITC-CD44 double-staining ( A ), ALDEFLUOR assay ( B ), or serially passaged mammosphere formation assay from which mammospheres were counted under a microscope by independent investigators ( C ). * Denotes that the number of mammospheres obtained from Akt-expressing cells differed significantly ( p < 0.05) from the vector controls.

    Journal: Molecular Cancer

    Article Title: Dichotomy effects of Akt signaling in breast cancer

    doi: 10.1186/1476-4598-11-61

    Figure Lengend Snippet: Activated Akt signaling decreases breast stem/progenitor cell subpopulations. MCF10A cells were infected with retroviruses to respectively express empty vector (Babe), Akt1, Akt2, or Akt3 and then subject to PE-CD24 and FITC-CD44 double-staining ( A ), ALDEFLUOR assay ( B ), or serially passaged mammosphere formation assay from which mammospheres were counted under a microscope by independent investigators ( C ). * Denotes that the number of mammospheres obtained from Akt-expressing cells differed significantly ( p < 0.05) from the vector controls.

    Article Snippet: Antibiotic-antimycotic (100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphoterincin) was routinely included in medium to prevent microbial contamination. pBabe-Puro, pBabe-Puro-Myr-Flag-Akt1 [ ], pBabe-Puro-Myr-HA-Akt2, pBabe-Puro-Myr-HA-Akt3, pBabe-Bleo, and pBabe-Bleo-IGF-1R, were purchased from Addgene Inc. To obtain infectious retrovirions prior to transducing Myr-Akt into target cells, retroviral vectors were first introduced into packaging cells known as Phoenix™ Ampho (Orbigen, Inc.) by a calcium phosphate transfection method.

    Techniques: Infection, Plasmid Preparation, Double Staining, Tube Formation Assay, Microscopy, Expressing

    The inhibitory effects of Akt signaling on EMT, cell motility and stem/progenitor cell subfraction can be attenuated in cells that become progressively malignant. A. MI, MII and MIII cells were infected with retroviruses expressing vector control (Babe), Akt1 (1), Akt2 (2), or Akt3 (3), followed by RT-qPCR analysis for assessing expression levels of EMT-associated transcripts. Heatmap represents relative mRNA levels of various transcripts in Akt-expressing cells compared to the ones in control cells set as 1. B. Infected MI, MII, or MIII cells were tested for migration assay 12 hours after seeding on the top chamber of transwell plate and * denotes a significant change ( p < 0.05) as compared to the control cells (Babe). C. Infected MI, MII or MIII cells were subjected to ALDEFLUOR staining (upper) or PE-CD24 plus FITC-CD44 double-staining (lower), followed by FACS analysis to quantify the frequency of ALDH + and CD44 +/high /CD24 -/low subpopulations. * denotes significant (p < 0.05) difference between the Akt-expressing cells and the counterpart control.

    Journal: Molecular Cancer

    Article Title: Dichotomy effects of Akt signaling in breast cancer

    doi: 10.1186/1476-4598-11-61

    Figure Lengend Snippet: The inhibitory effects of Akt signaling on EMT, cell motility and stem/progenitor cell subfraction can be attenuated in cells that become progressively malignant. A. MI, MII and MIII cells were infected with retroviruses expressing vector control (Babe), Akt1 (1), Akt2 (2), or Akt3 (3), followed by RT-qPCR analysis for assessing expression levels of EMT-associated transcripts. Heatmap represents relative mRNA levels of various transcripts in Akt-expressing cells compared to the ones in control cells set as 1. B. Infected MI, MII, or MIII cells were tested for migration assay 12 hours after seeding on the top chamber of transwell plate and * denotes a significant change ( p < 0.05) as compared to the control cells (Babe). C. Infected MI, MII or MIII cells were subjected to ALDEFLUOR staining (upper) or PE-CD24 plus FITC-CD44 double-staining (lower), followed by FACS analysis to quantify the frequency of ALDH + and CD44 +/high /CD24 -/low subpopulations. * denotes significant (p < 0.05) difference between the Akt-expressing cells and the counterpart control.

    Article Snippet: Antibiotic-antimycotic (100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphoterincin) was routinely included in medium to prevent microbial contamination. pBabe-Puro, pBabe-Puro-Myr-Flag-Akt1 [ ], pBabe-Puro-Myr-HA-Akt2, pBabe-Puro-Myr-HA-Akt3, pBabe-Bleo, and pBabe-Bleo-IGF-1R, were purchased from Addgene Inc. To obtain infectious retrovirions prior to transducing Myr-Akt into target cells, retroviral vectors were first introduced into packaging cells known as Phoenix™ Ampho (Orbigen, Inc.) by a calcium phosphate transfection method.

    Techniques: Infection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Migration, Staining, Double Staining

    Activated Akt signaling conveys resistance to chemotherapeutic drugs in an isoform redundant manner. MCF10A cells were infected with retroviruses to respectively express empty vector (Babe), Akt1, Akt2 or Akt3 and then treated with various dosages of paclitaxel ( A ) and doxorubicin ( B ) for 2 and 5 days. % of viable cells was calculated by setting untreated groups as 100% to which the drug treated cells were compared. Akt-overexpression significantly sustained cell viability in response to treatments by cytotoxic agents ( p < 0.05).

    Journal: Molecular Cancer

    Article Title: Dichotomy effects of Akt signaling in breast cancer

    doi: 10.1186/1476-4598-11-61

    Figure Lengend Snippet: Activated Akt signaling conveys resistance to chemotherapeutic drugs in an isoform redundant manner. MCF10A cells were infected with retroviruses to respectively express empty vector (Babe), Akt1, Akt2 or Akt3 and then treated with various dosages of paclitaxel ( A ) and doxorubicin ( B ) for 2 and 5 days. % of viable cells was calculated by setting untreated groups as 100% to which the drug treated cells were compared. Akt-overexpression significantly sustained cell viability in response to treatments by cytotoxic agents ( p < 0.05).

    Article Snippet: Antibiotic-antimycotic (100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphoterincin) was routinely included in medium to prevent microbial contamination. pBabe-Puro, pBabe-Puro-Myr-Flag-Akt1 [ ], pBabe-Puro-Myr-HA-Akt2, pBabe-Puro-Myr-HA-Akt3, pBabe-Bleo, and pBabe-Bleo-IGF-1R, were purchased from Addgene Inc. To obtain infectious retrovirions prior to transducing Myr-Akt into target cells, retroviral vectors were first introduced into packaging cells known as Phoenix™ Ampho (Orbigen, Inc.) by a calcium phosphate transfection method.

    Techniques: Infection, Plasmid Preparation, Over Expression

    AKT1 Phosphorylates HSF1 on S326 and induces HSF1 activity independent of mTORC1. (A-C) Indicated cell lines were transfected with vector, AKT1, AKT2 or AKT3 along with the HSE luciferase reporter for 48 h. Firefly/Renilla ratios were compared across groups. (D) Recombinant proteins were incubated at 30 °C for 2 h in the presence of ATP and subsequently subjected to SDS/PAGE and immunoblotting with indicated antibodies. (E) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cell lysates were subjected to SDS/PAGE and immunoblotting with indicated antibodies. (F-G) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 in the presence or absence of rapamycin (10–20 nM). Cell lysates were subjected to firefly reporter assay (F) or immunoblotting with indicated antibodies (G). Experiments were completed in triplicate and analysed using one-way ANOVA and Tukey’s post-hoc test. Data are presented as mean ± SEM. * P < 0.05.

    Journal: The FEBS journal

    Article Title: AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation

    doi: 10.1111/febs.16375

    Figure Lengend Snippet: AKT1 Phosphorylates HSF1 on S326 and induces HSF1 activity independent of mTORC1. (A-C) Indicated cell lines were transfected with vector, AKT1, AKT2 or AKT3 along with the HSE luciferase reporter for 48 h. Firefly/Renilla ratios were compared across groups. (D) Recombinant proteins were incubated at 30 °C for 2 h in the presence of ATP and subsequently subjected to SDS/PAGE and immunoblotting with indicated antibodies. (E) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cell lysates were subjected to SDS/PAGE and immunoblotting with indicated antibodies. (F-G) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 in the presence or absence of rapamycin (10–20 nM). Cell lysates were subjected to firefly reporter assay (F) or immunoblotting with indicated antibodies (G). Experiments were completed in triplicate and analysed using one-way ANOVA and Tukey’s post-hoc test. Data are presented as mean ± SEM. * P < 0.05.

    Article Snippet: The pcDNA3 Myr HA AKT3 plasmid was purchased from Addgene (ID 9017, RRID:Addgene_9017), which was originally established by Dr William Sellers.

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Recombinant, Incubation, SDS Page, Western Blot, Reporter Assay

    AKT1 promotes HSF1 trimerization and DNA binding. (A-B) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cells were fixed with 1 mM EGS for 30 min and lysates were subjected to SDS/PAGE and immunoblotting for HSF1 trimers (A) and AKT isoforms (B). (C) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cells were fixed with formaldehyde, lysed and sonicated prior to ChIP with either control IgG or HSF1 antibodies. ChIP product was subjected to qPCR for indicated gene promoters. (D-E) HEK-293 cells were transfected with either empty vector, AKT1, AKT2 or AKT3 for 48 h. Total RNA was collected from cells and subjected to RT-qPCR for the indicated genes. Experiments were completed in triplicate and analysed using one-way ANOVA and Tukey’s post-hoc test. Data are presented as mean ± SEM. * P < 0.05.

    Journal: The FEBS journal

    Article Title: AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation

    doi: 10.1111/febs.16375

    Figure Lengend Snippet: AKT1 promotes HSF1 trimerization and DNA binding. (A-B) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cells were fixed with 1 mM EGS for 30 min and lysates were subjected to SDS/PAGE and immunoblotting for HSF1 trimers (A) and AKT isoforms (B). (C) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cells were fixed with formaldehyde, lysed and sonicated prior to ChIP with either control IgG or HSF1 antibodies. ChIP product was subjected to qPCR for indicated gene promoters. (D-E) HEK-293 cells were transfected with either empty vector, AKT1, AKT2 or AKT3 for 48 h. Total RNA was collected from cells and subjected to RT-qPCR for the indicated genes. Experiments were completed in triplicate and analysed using one-way ANOVA and Tukey’s post-hoc test. Data are presented as mean ± SEM. * P < 0.05.

    Article Snippet: The pcDNA3 Myr HA AKT3 plasmid was purchased from Addgene (ID 9017, RRID:Addgene_9017), which was originally established by Dr William Sellers.

    Techniques: Binding Assay, Transfection, Plasmid Preparation, SDS Page, Western Blot, Sonication, Quantitative RT-PCR

    a Representation of the IL-3 signaling pathway in the murine Ba/F3 cell upon binding of IL-3. b General overview of the established cell lines Ba/F3 myr-Akt1 (gray), Ba/F3 myr-Akt2 (red), and Ba/F3 myr-Akt3 (yellow). Graph showing the seeded cell number of the Ba/F3 constructs and resulting luminescent signals following treatment with CTG solution compared to the parental system (dotted line, white). The experiments were performed in triplicates with n = 3. Error bars indicate s.d. Immunoblot of myr-Akt isoform systems highlighting the molecular expression levels, activities, and alterations of downstream targets of Akt ( n = 1 biologically independent experiment). c Addressing the Ba/F3 myr-Akt with CAAI borussertib to investigate the sensitivity of the established system. Structure of used inhibitor and schematic representation of the binding mode. Graph showing the remaining viable cells as a percentage with respect to the used concentration of borussertib. Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Immunoblots resolved individual Akt isoforms’ presence and activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). d Addressing the Ba/F3 myr-Akt cells with isoform-selective CAAIs 15a / 16b to investigate selectivity translation into the established system. Graph showing the remaining viable cells as a percentage with respect to the used concentration of 15a and 16b . Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Structure of used inhibitors. Immunoblots resolved isoform selective activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). In c , d the box plot middle line represents the mean value, the box represents the first and third quartile and the error bars indicate s.e. Source Data are provided with this paper.

    Journal: Nature Communications

    Article Title: Cellular model system to dissect the isoform-selectivity of Akt inhibitors

    doi: 10.1038/s41467-021-25512-8

    Figure Lengend Snippet: a Representation of the IL-3 signaling pathway in the murine Ba/F3 cell upon binding of IL-3. b General overview of the established cell lines Ba/F3 myr-Akt1 (gray), Ba/F3 myr-Akt2 (red), and Ba/F3 myr-Akt3 (yellow). Graph showing the seeded cell number of the Ba/F3 constructs and resulting luminescent signals following treatment with CTG solution compared to the parental system (dotted line, white). The experiments were performed in triplicates with n = 3. Error bars indicate s.d. Immunoblot of myr-Akt isoform systems highlighting the molecular expression levels, activities, and alterations of downstream targets of Akt ( n = 1 biologically independent experiment). c Addressing the Ba/F3 myr-Akt with CAAI borussertib to investigate the sensitivity of the established system. Structure of used inhibitor and schematic representation of the binding mode. Graph showing the remaining viable cells as a percentage with respect to the used concentration of borussertib. Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Immunoblots resolved individual Akt isoforms’ presence and activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). d Addressing the Ba/F3 myr-Akt cells with isoform-selective CAAIs 15a / 16b to investigate selectivity translation into the established system. Graph showing the remaining viable cells as a percentage with respect to the used concentration of 15a and 16b . Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Structure of used inhibitors. Immunoblots resolved isoform selective activity within the three model systems after 4-h treatment ( n = 1 biologically independent experiment). In c , d the box plot middle line represents the mean value, the box represents the first and third quartile and the error bars indicate s.e. Source Data are provided with this paper.

    Article Snippet: 1051 pBABEpuroL-AKT1, 1271 pBABEpuroL-Myr-HA-AKT2, and 1272 pBABEpuroL-Myr-HA-AKT3 were a gift from William Sellers (Addgene plasmid #9011/ 9018/ 9019; http://n2t.net/addgene:90011/ 9018/ 9019; RRID: Addgene_ 9011/ 9018/ 9019).

    Techniques: Binding Assay, Construct, Western Blot, Expressing, Concentration Assay, Activity Assay

    Biochemical evaluation of covalent-allosteric Akt inhibitors with the Akt isoforms.

    Journal: Nature Communications

    Article Title: Cellular model system to dissect the isoform-selectivity of Akt inhibitors

    doi: 10.1038/s41467-021-25512-8

    Figure Lengend Snippet: Biochemical evaluation of covalent-allosteric Akt inhibitors with the Akt isoforms.

    Article Snippet: 1051 pBABEpuroL-AKT1, 1271 pBABEpuroL-Myr-HA-AKT2, and 1272 pBABEpuroL-Myr-HA-AKT3 were a gift from William Sellers (Addgene plasmid #9011/ 9018/ 9019; http://n2t.net/addgene:90011/ 9018/ 9019; RRID: Addgene_ 9011/ 9018/ 9019).

    Techniques:

    a General overview of the closed conformation of protein kinase Akt (kinase domain: gray, PH domain: green; based on PDB: 6HHF) and the druggable binding pockets (ATP-pocket within kinase domain: gray (PDB: 4GV1) , allosteric-interdomain pocket: blue (PDB: 6HHF), PIP 3 -binding pocket within PH domain: gray (PDB 2UVM) ). The variety of the binding site forming residues among the three isoforms are given in percentage; b View of the interdomain binding site in Akt1 (white; PDB 6S9X: with the covalent-allosteric Akt inhibitor 15c (blue)) in comparison to homology models of Akt2 (red) and Akt3 (yellow). The structural changes within the allosteric site and the different side chain residues of each isoform are highlighted; c Modeling studies with recently found pyrazinonic CAAI 16c into Akt isoform homology model highlight structural differences and possible interactions within the allosteric binding site (Akt1(white) PDB: 6S9X; Akt2 (red) and Akt3 (yellow) homology model) . The side view shows an enlarged space in Akt2 and Akt3, ideally to allow the binding of the hydroxyl phenyl moiety (Supplementary Fig. ); d Structure-guided design approach towards a focused set of small molecules for evaluation of Akt isoform-selectivity: trisubstituted, biarylic pyridines function as core scaffold (blue). A benzimidazolone linker (green) is decorated with the Michael acceptor warhead (gray) to address the nucleophilic cysteine residues within the allosteric binding site. Different moieties (red) with various chemical properties and spatial demand should be connected over a defined linkage to the core molecule.

    Journal: Nature Communications

    Article Title: Cellular model system to dissect the isoform-selectivity of Akt inhibitors

    doi: 10.1038/s41467-021-25512-8

    Figure Lengend Snippet: a General overview of the closed conformation of protein kinase Akt (kinase domain: gray, PH domain: green; based on PDB: 6HHF) and the druggable binding pockets (ATP-pocket within kinase domain: gray (PDB: 4GV1) , allosteric-interdomain pocket: blue (PDB: 6HHF), PIP 3 -binding pocket within PH domain: gray (PDB 2UVM) ). The variety of the binding site forming residues among the three isoforms are given in percentage; b View of the interdomain binding site in Akt1 (white; PDB 6S9X: with the covalent-allosteric Akt inhibitor 15c (blue)) in comparison to homology models of Akt2 (red) and Akt3 (yellow). The structural changes within the allosteric site and the different side chain residues of each isoform are highlighted; c Modeling studies with recently found pyrazinonic CAAI 16c into Akt isoform homology model highlight structural differences and possible interactions within the allosteric binding site (Akt1(white) PDB: 6S9X; Akt2 (red) and Akt3 (yellow) homology model) . The side view shows an enlarged space in Akt2 and Akt3, ideally to allow the binding of the hydroxyl phenyl moiety (Supplementary Fig. ); d Structure-guided design approach towards a focused set of small molecules for evaluation of Akt isoform-selectivity: trisubstituted, biarylic pyridines function as core scaffold (blue). A benzimidazolone linker (green) is decorated with the Michael acceptor warhead (gray) to address the nucleophilic cysteine residues within the allosteric binding site. Different moieties (red) with various chemical properties and spatial demand should be connected over a defined linkage to the core molecule.

    Article Snippet: 1051 pBABEpuroL-AKT1, 1271 pBABEpuroL-Myr-HA-AKT2, and 1272 pBABEpuroL-Myr-HA-AKT3 were a gift from William Sellers (Addgene plasmid #9011/ 9018/ 9019; http://n2t.net/addgene:90011/ 9018/ 9019; RRID: Addgene_ 9011/ 9018/ 9019).

    Techniques: Binding Assay

    Kinetic evaluation of covalent-allosteric Akt ligands with the three Akt isoforms.

    Journal: Nature Communications

    Article Title: Cellular model system to dissect the isoform-selectivity of Akt inhibitors

    doi: 10.1038/s41467-021-25512-8

    Figure Lengend Snippet: Kinetic evaluation of covalent-allosteric Akt ligands with the three Akt isoforms.

    Article Snippet: 1051 pBABEpuroL-AKT1, 1271 pBABEpuroL-Myr-HA-AKT2, and 1272 pBABEpuroL-Myr-HA-AKT3 were a gift from William Sellers (Addgene plasmid #9011/ 9018/ 9019; http://n2t.net/addgene:90011/ 9018/ 9019; RRID: Addgene_ 9011/ 9018/ 9019).

    Techniques:

    Akt family members influence GR- and DEX-mediated transactivation of the IEtu1 collapsed promoter. (A) Neuro-2A cells were transfected with the IEtu1 collapsed promoter construct containing the firefly luciferase reporter gene (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1, Akt2, or Akt3 (1.0 μg). (B) Neuro-2A cells were transfected with the IEtu1 collapsed promoter (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg). or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped fetal bovine serum (FBS) at approximately 24 h after transfection, and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments, and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency

    doi: 10.1128/JVI.00901-20

    Figure Lengend Snippet: Akt family members influence GR- and DEX-mediated transactivation of the IEtu1 collapsed promoter. (A) Neuro-2A cells were transfected with the IEtu1 collapsed promoter construct containing the firefly luciferase reporter gene (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1, Akt2, or Akt3 (1.0 μg). (B) Neuro-2A cells were transfected with the IEtu1 collapsed promoter (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg). or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped fetal bovine serum (FBS) at approximately 24 h after transfection, and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments, and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.

    Article Snippet: A plasmid that expresses Akt3 was a gift from William Sellers (1236 pcDNA3 Myr HA Akt3, plasmid 9017; Addgene).

    Techniques: Transfection, Construct, Luciferase, Mutagenesis, Plasmid Preparation, Incubation

    Akt1 and Akt2 impair GR-mediated activation of the MMTV-LTR promoter. (A) Neuro-2A cells were transfected with the MMTV-LTR promoter construct (0.5 μg) and, where indicated, plasmids that express GR (1.0 μg), Akt1, Akt2, or Akt3 (1.0 μg). (B) Neuro-2A cells were transfected with the MMTV-LTR promoter construct (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg), or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped FBS at approximately 24 h after transfection and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency

    doi: 10.1128/JVI.00901-20

    Figure Lengend Snippet: Akt1 and Akt2 impair GR-mediated activation of the MMTV-LTR promoter. (A) Neuro-2A cells were transfected with the MMTV-LTR promoter construct (0.5 μg) and, where indicated, plasmids that express GR (1.0 μg), Akt1, Akt2, or Akt3 (1.0 μg). (B) Neuro-2A cells were transfected with the MMTV-LTR promoter construct (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg), or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped FBS at approximately 24 h after transfection and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.

    Article Snippet: A plasmid that expresses Akt3 was a gift from William Sellers (1236 pcDNA3 Myr HA Akt3, plasmid 9017; Addgene).

    Techniques: Activation Assay, Transfection, Construct, Mutagenesis, Plasmid Preparation, Luciferase, Incubation

    Akt3 efficiently promotes neurite formation in Neuro-2A cells. Neuro-2A cells were cotransfected with an empty vector (pcDNA3.1) (A), a plasmid expressing Akt1 or Akt2 (Panel B), or Akt3 (C and D) (1 μg plasmid DNA) and a plasmid expressing the lacZ gene (0.1 μg plasmid) to mark transfected cells. (B) A typical result from cells transfected with Akt1 or Akt2. To induce neurite sprouting, 24 h after transfection, cells were seeded into new plates at a low density (2,000 cells/cm2) and then incubated with minimal essential medium (MEM) that contained 0.5% serum for 3 days. Cells were fixed, and β-Gal+ cells were detected by staining.

    Journal: Journal of Virology

    Article Title: Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency

    doi: 10.1128/JVI.00901-20

    Figure Lengend Snippet: Akt3 efficiently promotes neurite formation in Neuro-2A cells. Neuro-2A cells were cotransfected with an empty vector (pcDNA3.1) (A), a plasmid expressing Akt1 or Akt2 (Panel B), or Akt3 (C and D) (1 μg plasmid DNA) and a plasmid expressing the lacZ gene (0.1 μg plasmid) to mark transfected cells. (B) A typical result from cells transfected with Akt1 or Akt2. To induce neurite sprouting, 24 h after transfection, cells were seeded into new plates at a low density (2,000 cells/cm2) and then incubated with minimal essential medium (MEM) that contained 0.5% serum for 3 days. Cells were fixed, and β-Gal+ cells were detected by staining.

    Article Snippet: A plasmid that expresses Akt3 was a gift from William Sellers (1236 pcDNA3 Myr HA Akt3, plasmid 9017; Addgene).

    Techniques: Plasmid Preparation, Expressing, Transfection, Incubation, Staining

    Akt3, but not Akt1or Akt2, significantly increased neurite formation in Neuro-2A cells. The relative efficiency of β-Gal+ cells containing neurites was calculated by dividing the number of β-Gal+ cells with a neurite length at least twice the diameter of the cell by the total number of β-Gal+ cells. The % of β-Gal+ cells with neurites in the control was set at 1. The other samples were compared to the control to obtain the relative efficiency of neurite formation. The average of three independent experiments is shown with the respective standard deviation. An asterisk denotes significant differences (P < 0.05) in β-Gal+ Neuro-2A cells containing neurites following transfection with the Akt family member relative to the number of β-Gal+ Neuro-2A cells with neurites following transfection with an empty vector, as determined by the one-way analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) multiple means comparison tests.

    Journal: Journal of Virology

    Article Title: Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency

    doi: 10.1128/JVI.00901-20

    Figure Lengend Snippet: Akt3, but not Akt1or Akt2, significantly increased neurite formation in Neuro-2A cells. The relative efficiency of β-Gal+ cells containing neurites was calculated by dividing the number of β-Gal+ cells with a neurite length at least twice the diameter of the cell by the total number of β-Gal+ cells. The % of β-Gal+ cells with neurites in the control was set at 1. The other samples were compared to the control to obtain the relative efficiency of neurite formation. The average of three independent experiments is shown with the respective standard deviation. An asterisk denotes significant differences (P < 0.05) in β-Gal+ Neuro-2A cells containing neurites following transfection with the Akt family member relative to the number of β-Gal+ Neuro-2A cells with neurites following transfection with an empty vector, as determined by the one-way analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) multiple means comparison tests.

    Article Snippet: A plasmid that expresses Akt3 was a gift from William Sellers (1236 pcDNA3 Myr HA Akt3, plasmid 9017; Addgene).

    Techniques: Standard Deviation, Transfection, Plasmid Preparation