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    Name:
    Akt Control Cell Extracts
    Description:
    Phosphorylated Akt Cell Extracts Total cell extracts from Jurkat cells serum starved overnight and then treated with Calyculin A CST 9902 to preserve their activated Akt state serve as a positive control Supplied in SDS Sample Buffer
    Catalog Number:
    9273
    Price:
    None
    Applications:
    Western Blot
    Category:
    Experimental Controls
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc akt
    HGF and SCF preserved the mitochondrial function in hBMSCs via the <t>PI3K/AKT,</t> ERK1/2, and STAT3 signaling pathway. a Western blot results of the expression of mitochondrial-relative proteins (Mfn1, Mfn2, SOD2, and Catalase) and the PI3K/AKT, Erk1/2, and STAT3 signaling pathway-related proteins in passage 3 (P3) and passage 8 (P8) hBMSCs cultured in SHED-CM (P8-SHED-CM) and treated with 100 ng/ml (P8-HGF 100), 10 ng/ml SCF (P8-SCF 10), and the combination of 100 ng/ml HGF and 10 ng/ml SCF (P8-H+S). b Relative expression levels of the protein showed in the Western blot. ns, not significant. c Western blot results of the expression of mitochondrial oxidative stress-related proteins, Catalase and SOD2, and the PI3K/AKT signaling pathway-related proteins in P3 hBMSCs treated with 100 ng/ml HGF, 10 μM LY294002 plus 100 ng/ml HGF, 10 ng/ml SCF, and 10 μM LY294002 plus 10 ng/ml SCF. d Western blot results of the expression of mitochondrial oxidative stress-related proteins, Catalase and SOD2, and the PI3K/AKT signaling pathway-related proteins in P3 hBMSCs treated with 100 ng/ml HGF, 10 μM U0126 plus 100 ng/ml HGF, 10 ng/ml SCF, and 10 μM U0126 plus 10 ng/ml SCF. e Western blot results of the expression of mitochondrial oxidative stress-related proteins, Catalase and SOD2, and the PI3K/AKT signaling pathway-related proteins in P3 hBMSCs treated with 100 ng/ml HGF, 5 μM Stattic plus 100 ng/ml HGF, 10 ng/ml SCF, and 10 μM Stattic plus 10 ng/ml SCF. *P
    Phosphorylated Akt Cell Extracts Total cell extracts from Jurkat cells serum starved overnight and then treated with Calyculin A CST 9902 to preserve their activated Akt state serve as a positive control Supplied in SDS Sample Buffer
    https://www.bioz.com/result/akt/product/Cell Signaling Technology Inc
    Average 99 stars, based on 7750 article reviews
    Price from $9.99 to $1999.99
    akt - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Hepatocyte growth factor (HGF) and stem cell factor (SCF) maintained the stemness of human bone marrow mesenchymal stem cells (hBMSCs) during long-term expansion by preserving mitochondrial function via the PI3K/AKT, ERK1/2, and STAT3 signaling pathways"

    Article Title: Hepatocyte growth factor (HGF) and stem cell factor (SCF) maintained the stemness of human bone marrow mesenchymal stem cells (hBMSCs) during long-term expansion by preserving mitochondrial function via the PI3K/AKT, ERK1/2, and STAT3 signaling pathways

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-020-01830-4

    HGF and SCF preserved the mitochondrial function in hBMSCs via the PI3K/AKT, ERK1/2, and STAT3 signaling pathway. a Western blot results of the expression of mitochondrial-relative proteins (Mfn1, Mfn2, SOD2, and Catalase) and the PI3K/AKT, Erk1/2, and STAT3 signaling pathway-related proteins in passage 3 (P3) and passage 8 (P8) hBMSCs cultured in SHED-CM (P8-SHED-CM) and treated with 100 ng/ml (P8-HGF 100), 10 ng/ml SCF (P8-SCF 10), and the combination of 100 ng/ml HGF and 10 ng/ml SCF (P8-H+S). b Relative expression levels of the protein showed in the Western blot. ns, not significant. c Western blot results of the expression of mitochondrial oxidative stress-related proteins, Catalase and SOD2, and the PI3K/AKT signaling pathway-related proteins in P3 hBMSCs treated with 100 ng/ml HGF, 10 μM LY294002 plus 100 ng/ml HGF, 10 ng/ml SCF, and 10 μM LY294002 plus 10 ng/ml SCF. d Western blot results of the expression of mitochondrial oxidative stress-related proteins, Catalase and SOD2, and the PI3K/AKT signaling pathway-related proteins in P3 hBMSCs treated with 100 ng/ml HGF, 10 μM U0126 plus 100 ng/ml HGF, 10 ng/ml SCF, and 10 μM U0126 plus 10 ng/ml SCF. e Western blot results of the expression of mitochondrial oxidative stress-related proteins, Catalase and SOD2, and the PI3K/AKT signaling pathway-related proteins in P3 hBMSCs treated with 100 ng/ml HGF, 5 μM Stattic plus 100 ng/ml HGF, 10 ng/ml SCF, and 10 μM Stattic plus 10 ng/ml SCF. *P
    Figure Legend Snippet: HGF and SCF preserved the mitochondrial function in hBMSCs via the PI3K/AKT, ERK1/2, and STAT3 signaling pathway. a Western blot results of the expression of mitochondrial-relative proteins (Mfn1, Mfn2, SOD2, and Catalase) and the PI3K/AKT, Erk1/2, and STAT3 signaling pathway-related proteins in passage 3 (P3) and passage 8 (P8) hBMSCs cultured in SHED-CM (P8-SHED-CM) and treated with 100 ng/ml (P8-HGF 100), 10 ng/ml SCF (P8-SCF 10), and the combination of 100 ng/ml HGF and 10 ng/ml SCF (P8-H+S). b Relative expression levels of the protein showed in the Western blot. ns, not significant. c Western blot results of the expression of mitochondrial oxidative stress-related proteins, Catalase and SOD2, and the PI3K/AKT signaling pathway-related proteins in P3 hBMSCs treated with 100 ng/ml HGF, 10 μM LY294002 plus 100 ng/ml HGF, 10 ng/ml SCF, and 10 μM LY294002 plus 10 ng/ml SCF. d Western blot results of the expression of mitochondrial oxidative stress-related proteins, Catalase and SOD2, and the PI3K/AKT signaling pathway-related proteins in P3 hBMSCs treated with 100 ng/ml HGF, 10 μM U0126 plus 100 ng/ml HGF, 10 ng/ml SCF, and 10 μM U0126 plus 10 ng/ml SCF. e Western blot results of the expression of mitochondrial oxidative stress-related proteins, Catalase and SOD2, and the PI3K/AKT signaling pathway-related proteins in P3 hBMSCs treated with 100 ng/ml HGF, 5 μM Stattic plus 100 ng/ml HGF, 10 ng/ml SCF, and 10 μM Stattic plus 10 ng/ml SCF. *P

    Techniques Used: Western Blot, Expressing, Cell Culture

    2) Product Images from "An engineered Abcb4 expressing model reveals the central role of NF-κB in the regulation of drug resistance in zebrafish"

    Article Title: An engineered Abcb4 expressing model reveals the central role of NF-κB in the regulation of drug resistance in zebrafish

    Journal: bioRxiv

    doi: 10.1101/2020.03.30.016824

    DOX and VCR regulate the expression of abcb4 through the AKT/NF-κB and ERK/NF-κB signaling pathways in Tg(abcb4:EGFP) zebrafish embryos. (A) The embryos were treated with 6.5 μM LY294002 and/or 2.5 μM U0126 inhibitor prior to 15 μM DOX, then the level of AKT/p-AKT, ERK1/2, p-ERK1/2, P65/p-P65, abcb4, and EGFP was detected and the images were photographed. (B) The level p-AKT, p-ERK1/2, p-P65, abcb4, and EGFP was analyzed by data normalization with β-actin antibody. Compared to control group, the high expression of abcb4 and EGFP presented in DOX, DOX+LY294002, and DOX+U0126 groups, whilst abcb4 was downregulated in DOX + LY294002 + U0126 group (* P
    Figure Legend Snippet: DOX and VCR regulate the expression of abcb4 through the AKT/NF-κB and ERK/NF-κB signaling pathways in Tg(abcb4:EGFP) zebrafish embryos. (A) The embryos were treated with 6.5 μM LY294002 and/or 2.5 μM U0126 inhibitor prior to 15 μM DOX, then the level of AKT/p-AKT, ERK1/2, p-ERK1/2, P65/p-P65, abcb4, and EGFP was detected and the images were photographed. (B) The level p-AKT, p-ERK1/2, p-P65, abcb4, and EGFP was analyzed by data normalization with β-actin antibody. Compared to control group, the high expression of abcb4 and EGFP presented in DOX, DOX+LY294002, and DOX+U0126 groups, whilst abcb4 was downregulated in DOX + LY294002 + U0126 group (* P

    Techniques Used: Expressing

    3) Product Images from "ARHGEF3 regulates skeletal muscle regeneration and strength through autophagy"

    Article Title: ARHGEF3 regulates skeletal muscle regeneration and strength through autophagy

    Journal: bioRxiv

    doi: 10.1101/2020.02.28.970756

    ARHGEF3 regulation of muscle regeneration is independent of mTORC2-Akt signaling. TA muscles from 3-month-old WT and ARHGEF3 KO (AKO) male mice were injected with BaCl 2 (injury) or saline (uninjured control, 0 day). ( A ) The muscles were collected 7 and 14 days after injury (AI) and analyzed by Western blotting for phosphorylated (p)/total Akt ratio and GAPDH protein expression ( n = 5-6). ( B - C ) Mice were treated with vehicle (DMSO) or triciribine (TCB) upon injury. Muscle weight (MW) and maximal isometric tetanic force (Po) were measured 21 days AI and presented as % of contralateral uninjured control ( B , n = 4-9). Phosphorylated (P)/total (T) protein ratio for Akt and GSK3β was determined by Western blotting in injured AKO muscles collected 21 days AI ( C , n = 4-5). Data are presented as mean ± SEM. * P
    Figure Legend Snippet: ARHGEF3 regulation of muscle regeneration is independent of mTORC2-Akt signaling. TA muscles from 3-month-old WT and ARHGEF3 KO (AKO) male mice were injected with BaCl 2 (injury) or saline (uninjured control, 0 day). ( A ) The muscles were collected 7 and 14 days after injury (AI) and analyzed by Western blotting for phosphorylated (p)/total Akt ratio and GAPDH protein expression ( n = 5-6). ( B - C ) Mice were treated with vehicle (DMSO) or triciribine (TCB) upon injury. Muscle weight (MW) and maximal isometric tetanic force (Po) were measured 21 days AI and presented as % of contralateral uninjured control ( B , n = 4-9). Phosphorylated (P)/total (T) protein ratio for Akt and GSK3β was determined by Western blotting in injured AKO muscles collected 21 days AI ( C , n = 4-5). Data are presented as mean ± SEM. * P

    Techniques Used: Mouse Assay, Injection, Western Blot, Expressing

    The GEF activity of ARHGEF3 is critical for the regulation of muscle regeneration. ( A ) TA muscles from 3-month-old WT and ARHGEF3 KO (AKO) male mice were injected with BaCl 2 , collected 14 days after injury, and analyzed by Western blotting for active (A)/total (T) RhoA ratio ( n = 5-6). ( B ) TA muscles from ARHGEF3 KO (AKO) male mice were injected with BaCl 2 (injury) or saline (uninjured control, 0 day), and either collected 3 and 10 days (d) after injury (AI) for analysis of F4/80 and GAPDH protein expression by Western blotting (upper panel, n = 3) or transfected with plasmids encoding WT- or L269E-ARHGEF3 10 days AI and allowed to recovery until 21 days AI (lower panel). ( C ) Active/total RhoA ratio, phosphorylated (p)/total Akt ratio, and GAPDH protein expression were determined by Western blotting in the transfected injured muscles ( n = 3). ( D ) Muscle weight (MW) and maximal isometric tetanic force (Po) were measured in the transfected injured muscles and presented as % of contralateral uninjured control ( n = 3). Data are presented as mean ± SEM. * P
    Figure Legend Snippet: The GEF activity of ARHGEF3 is critical for the regulation of muscle regeneration. ( A ) TA muscles from 3-month-old WT and ARHGEF3 KO (AKO) male mice were injected with BaCl 2 , collected 14 days after injury, and analyzed by Western blotting for active (A)/total (T) RhoA ratio ( n = 5-6). ( B ) TA muscles from ARHGEF3 KO (AKO) male mice were injected with BaCl 2 (injury) or saline (uninjured control, 0 day), and either collected 3 and 10 days (d) after injury (AI) for analysis of F4/80 and GAPDH protein expression by Western blotting (upper panel, n = 3) or transfected with plasmids encoding WT- or L269E-ARHGEF3 10 days AI and allowed to recovery until 21 days AI (lower panel). ( C ) Active/total RhoA ratio, phosphorylated (p)/total Akt ratio, and GAPDH protein expression were determined by Western blotting in the transfected injured muscles ( n = 3). ( D ) Muscle weight (MW) and maximal isometric tetanic force (Po) were measured in the transfected injured muscles and presented as % of contralateral uninjured control ( n = 3). Data are presented as mean ± SEM. * P

    Techniques Used: Activity Assay, Mouse Assay, Injection, Western Blot, Expressing, Transfection

    4) Product Images from "HSP90 overexpression potentiates the B-cell receptor and fibroblast growth factor receptor survival signals in chronic lymphocytic leukemia cells"

    Article Title: HSP90 overexpression potentiates the B-cell receptor and fibroblast growth factor receptor survival signals in chronic lymphocytic leukemia cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.27409

    Pharmacologic inhibition or partial depletion of HSP90 in CLL cells reduces the levels of BCR signal mediators. ( A – C ) Impact of HSP90 inhibition on BCR signal mediators. Purified CLL cells were treated with a high-affinity HSP90 inhibitor, AUY922, at 0.2 µM dose or DMSO (vehicle control) for 24 hours. Cell lysates were analyzed for the expression of various signal mediators of the BCR pathway in western blots using specific antibody to LYN, SYK, BTK or AKT (A). Expression status of CD79a, BCAP, and PLCγ2 was also analyzed in the same CLL cell lysates used above in western blots using specific antibodies (B). Endogenous expression levels of CD19, ERK1/2 or STAT3 which are not reported to be the HSP90 client proteins were also analyzed in the same CLL cell lysates used above (C). However, phosphorylation status of ERK1/2 was examined as a downstream target of BCR signal in these cell lysates (C). GAPDH was used as loading control. CLL patients (P1, P4, P5) are indicated by numbers. ( D ) Targeted depletion of HSP90 reduces the levels of CD79a, BCAP, PLCγ2 and phosphorylation of ERK1/2 in CLL cells. Purified CLL cells from three previously untreated CLL patients (P16 – P18) were transduced with lentivirus expressing a HSP90-specific shRNA (indicated by “+”) or scrambled shRNA (indicated by “-”) for 24 hours. Cell lysates were prepared and analyzed for the expression of HSP90, CD79a, BCAP, PLCγ2 and P-ERK1/2 in western blots using specific antibodies. The blot of P-ERK1/2 was stripped and reprobed with an antibody to total ERK1/2. GAPDH was used as loading control. Level of HSP90 depletion in HSP90-shRNA transduced CLL cells was determined by densitometric quantification and presented as relative values of HSP90: GAPDH with respective to the scrambled shRNA transduced cells.
    Figure Legend Snippet: Pharmacologic inhibition or partial depletion of HSP90 in CLL cells reduces the levels of BCR signal mediators. ( A – C ) Impact of HSP90 inhibition on BCR signal mediators. Purified CLL cells were treated with a high-affinity HSP90 inhibitor, AUY922, at 0.2 µM dose or DMSO (vehicle control) for 24 hours. Cell lysates were analyzed for the expression of various signal mediators of the BCR pathway in western blots using specific antibody to LYN, SYK, BTK or AKT (A). Expression status of CD79a, BCAP, and PLCγ2 was also analyzed in the same CLL cell lysates used above in western blots using specific antibodies (B). Endogenous expression levels of CD19, ERK1/2 or STAT3 which are not reported to be the HSP90 client proteins were also analyzed in the same CLL cell lysates used above (C). However, phosphorylation status of ERK1/2 was examined as a downstream target of BCR signal in these cell lysates (C). GAPDH was used as loading control. CLL patients (P1, P4, P5) are indicated by numbers. ( D ) Targeted depletion of HSP90 reduces the levels of CD79a, BCAP, PLCγ2 and phosphorylation of ERK1/2 in CLL cells. Purified CLL cells from three previously untreated CLL patients (P16 – P18) were transduced with lentivirus expressing a HSP90-specific shRNA (indicated by “+”) or scrambled shRNA (indicated by “-”) for 24 hours. Cell lysates were prepared and analyzed for the expression of HSP90, CD79a, BCAP, PLCγ2 and P-ERK1/2 in western blots using specific antibodies. The blot of P-ERK1/2 was stripped and reprobed with an antibody to total ERK1/2. GAPDH was used as loading control. Level of HSP90 depletion in HSP90-shRNA transduced CLL cells was determined by densitometric quantification and presented as relative values of HSP90: GAPDH with respective to the scrambled shRNA transduced cells.

    Techniques Used: Inhibition, Purification, Expressing, Western Blot, Transduction, shRNA

    HSP90 forms a multi-molecular complex with kinases, lipase and adaptor molecule of the BCR pathway. HSP90 was immunoprecipitated from CLL cell lysates from previously untreated CLL patients (P1, P3, P5), followed by detection of CD79a, BCAP, and PLCγ2 ( A ) in western blots using specific antibodies. Similarly, CD79a, BCAP or PLCγ2 was immunoprecipitated individually from the same CLL cell lysates (P1, P3, P5) used above and the immunecomplex was analyzed for the presence of HSP90 in western blot using a specific antibody to HSP90 ( B ). HSP90 was further pulled down from the same CLL cell lysates (P1, P3, P5) used above to detect co-precipitation of LYN, SYK, BTK or AKT by western blot analyses using specific antibodies ( C ). CLL B-cell lysates from another CLL patient (P4) was also included in panel C. Immunoglobulin G heavy chain (IgG HC) was used as loading control.
    Figure Legend Snippet: HSP90 forms a multi-molecular complex with kinases, lipase and adaptor molecule of the BCR pathway. HSP90 was immunoprecipitated from CLL cell lysates from previously untreated CLL patients (P1, P3, P5), followed by detection of CD79a, BCAP, and PLCγ2 ( A ) in western blots using specific antibodies. Similarly, CD79a, BCAP or PLCγ2 was immunoprecipitated individually from the same CLL cell lysates (P1, P3, P5) used above and the immunecomplex was analyzed for the presence of HSP90 in western blot using a specific antibody to HSP90 ( B ). HSP90 was further pulled down from the same CLL cell lysates (P1, P3, P5) used above to detect co-precipitation of LYN, SYK, BTK or AKT by western blot analyses using specific antibodies ( C ). CLL B-cell lysates from another CLL patient (P4) was also included in panel C. Immunoglobulin G heavy chain (IgG HC) was used as loading control.

    Techniques Used: Immunoprecipitation, Western Blot

    5) Product Images from "Role of the E3 ubiquitin ligase RNF157 as a novel downstream effector linking PI3K and MAPK signaling pathways to the cell cycle"

    Article Title: Role of the E3 ubiquitin ligase RNF157 as a novel downstream effector linking PI3K and MAPK signaling pathways to the cell cycle

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.792754

    CDK2 promotes RNF157 phosphorylation. A , Western blot of FLAG-RNF157 co-immunoprecipitated with Myc-CDK2 from 624MEL melanoma cells transfected with the indicated vector combinations. B , analysis of the phosphorylation levels of FLAG-RNF157 by using the phosphospecific RNF157 antibody (pRNF157 S660–663 ) upon treatment with different inhibitors. RNF157 was co-transfected with control vector or Myc-CDK2 and where indicated was treated with DMSO as a control, CDK2 inhibitors CDK2i III (4.2 μ m ) and roscovitine (20 μ m ), or PI3K inhibitor/MEK inhibitor ( PI3Ki/MEKi ) in combination ( Combo ) for 6 h. C , HeLa cells were transfected with FLAG-RNF157 and serum-starved at 24 h post-transfection for 16 h. The cells were then treated with the inhibitors as indicated and lysed after EGF treatment (100 ng/ml for 5 min). SS lane , serum-starved unstimulated cells. Phosphorylation of RNF157 was analyzed by immunoblotting with the phosphospecific antibody. D , lysates of HeLa cells transfected with wild-type RNF157 or different phosphomutant plasmids together with control vector ( EV ) or Myc-CDK2 vector. Immunoblotting was performed with pRNF157 S660–663 , FLAG, Myc, and actin antibodies. E , lysates of HeLa cells transfected with FLAG-RNF157 and Myc-CDH1 expression plasmids and treated with the indicated inhibitors were subjected to immunoprecipitation with the Myc antibody followed by immunoblotting with FLAG, Myc, pRNF157 S660–663 , pRNF157 T170 , phospho-ERK ( pERK ), and actin antibodies. CDK2 inhibitor was used in two different concentrations, 2.1 and 4.2 μ m , for 8 h. F , lysates of HeLa cells transfected with control vector ( EV ), FLAG-RNF157, FLAG-RNF157(4SA), and Myc-CDH1 expression plasmids were subjected to immunoprecipitation with the Myc antibody followed by immunoblotting with FLAG, Myc, and actin antibodies. IP , immunoprecipitation, CoIP , co-immunoprecipitation; pAkt , phospho-AKT; pERK1/2 , phospho-ERK1/2; pCDK2 , phospho-CDK2.
    Figure Legend Snippet: CDK2 promotes RNF157 phosphorylation. A , Western blot of FLAG-RNF157 co-immunoprecipitated with Myc-CDK2 from 624MEL melanoma cells transfected with the indicated vector combinations. B , analysis of the phosphorylation levels of FLAG-RNF157 by using the phosphospecific RNF157 antibody (pRNF157 S660–663 ) upon treatment with different inhibitors. RNF157 was co-transfected with control vector or Myc-CDK2 and where indicated was treated with DMSO as a control, CDK2 inhibitors CDK2i III (4.2 μ m ) and roscovitine (20 μ m ), or PI3K inhibitor/MEK inhibitor ( PI3Ki/MEKi ) in combination ( Combo ) for 6 h. C , HeLa cells were transfected with FLAG-RNF157 and serum-starved at 24 h post-transfection for 16 h. The cells were then treated with the inhibitors as indicated and lysed after EGF treatment (100 ng/ml for 5 min). SS lane , serum-starved unstimulated cells. Phosphorylation of RNF157 was analyzed by immunoblotting with the phosphospecific antibody. D , lysates of HeLa cells transfected with wild-type RNF157 or different phosphomutant plasmids together with control vector ( EV ) or Myc-CDK2 vector. Immunoblotting was performed with pRNF157 S660–663 , FLAG, Myc, and actin antibodies. E , lysates of HeLa cells transfected with FLAG-RNF157 and Myc-CDH1 expression plasmids and treated with the indicated inhibitors were subjected to immunoprecipitation with the Myc antibody followed by immunoblotting with FLAG, Myc, pRNF157 S660–663 , pRNF157 T170 , phospho-ERK ( pERK ), and actin antibodies. CDK2 inhibitor was used in two different concentrations, 2.1 and 4.2 μ m , for 8 h. F , lysates of HeLa cells transfected with control vector ( EV ), FLAG-RNF157, FLAG-RNF157(4SA), and Myc-CDH1 expression plasmids were subjected to immunoprecipitation with the Myc antibody followed by immunoblotting with FLAG, Myc, and actin antibodies. IP , immunoprecipitation, CoIP , co-immunoprecipitation; pAkt , phospho-AKT; pERK1/2 , phospho-ERK1/2; pCDK2 , phospho-CDK2.

    Techniques Used: Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Co-Immunoprecipitation Assay

    6) Product Images from "OSU-CG5, a novel energy restriction mimetic agent, targets human colorectal cancer cells in vitro"

    Article Title: OSU-CG5, a novel energy restriction mimetic agent, targets human colorectal cancer cells in vitro

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2013.183

    The antiproliferative activity of OSU-CG5 in CRC cells is associated with energy restriction cellular responses. Western blot analysis of the expression levels of p-Akt, p-AMPK, β-TrCP, Sp1, Cyclin D1, p-mTOR, and p-p70S6K in HCT-116 cells after
    Figure Legend Snippet: The antiproliferative activity of OSU-CG5 in CRC cells is associated with energy restriction cellular responses. Western blot analysis of the expression levels of p-Akt, p-AMPK, β-TrCP, Sp1, Cyclin D1, p-mTOR, and p-p70S6K in HCT-116 cells after

    Techniques Used: Activity Assay, Western Blot, Expressing

    7) Product Images from "Muscle Mechanics and Ventricular Function: Structural and functional cardiac profile after prolonged duration of mechanical unloading: potential implications for myocardial recovery"

    Article Title: Muscle Mechanics and Ventricular Function: Structural and functional cardiac profile after prolonged duration of mechanical unloading: potential implications for myocardial recovery

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00187.2018

    Stress- and growth-related proteins and left ventricular assist device (LVAD) support. A−G : total and phosphorylated (p-)ERK42 ( A ), total and p-ERK44 ( B ), total and p-p38 MAPK ( C ), total and p-Akt ( D ), total and p-mechanistic target of rapamycin (mTOR; E ), corticotropin-releasing factor 1 (CRF1; F ), and CRF2 ( G ) in normal hearts ( n = 4) and samples from failing hearts before (pre-LVAD; n = 15) and after (post-LVAD; n = 15) LVAD implantation with representative blots. Molecular variables were compared using one-way ANOVA with post hoc Bonferroni analysis. Simple linear regression analysis was used to determine the effect of LVAD time as a continuous variable in clinical and molecular measurements. A t -test was used to compare the effect on LVAD duration as a categorical variable in the change in molecular measurement with LVAD. * P
    Figure Legend Snippet: Stress- and growth-related proteins and left ventricular assist device (LVAD) support. A−G : total and phosphorylated (p-)ERK42 ( A ), total and p-ERK44 ( B ), total and p-p38 MAPK ( C ), total and p-Akt ( D ), total and p-mechanistic target of rapamycin (mTOR; E ), corticotropin-releasing factor 1 (CRF1; F ), and CRF2 ( G ) in normal hearts ( n = 4) and samples from failing hearts before (pre-LVAD; n = 15) and after (post-LVAD; n = 15) LVAD implantation with representative blots. Molecular variables were compared using one-way ANOVA with post hoc Bonferroni analysis. Simple linear regression analysis was used to determine the effect of LVAD time as a continuous variable in clinical and molecular measurements. A t -test was used to compare the effect on LVAD duration as a categorical variable in the change in molecular measurement with LVAD. * P

    Techniques Used:

    8) Product Images from "Up-Regulated CCDC34 Contributes to the Proliferation and Metastasis of Hepatocellular Carcinoma"

    Article Title: Up-Regulated CCDC34 Contributes to the Proliferation and Metastasis of Hepatocellular Carcinoma

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S237399

    The activation of AKT pathways and EMT process hampered by silencing of CCDC34. Notes: ( A ) A scatter plot shows the number of the DEGs analyzed by the RNA sequencing in the BEL-7404-CCDC34-OE cells (fold change > 2.0 and P-value
    Figure Legend Snippet: The activation of AKT pathways and EMT process hampered by silencing of CCDC34. Notes: ( A ) A scatter plot shows the number of the DEGs analyzed by the RNA sequencing in the BEL-7404-CCDC34-OE cells (fold change > 2.0 and P-value

    Techniques Used: Activation Assay, RNA Sequencing Assay

    9) Product Images from "mTOR attenuates the inflammatory response in cardiomyocytes and prevents cardiac dysfunction in pathological hypertrophy"

    Article Title: mTOR attenuates the inflammatory response in cardiomyocytes and prevents cardiac dysfunction in pathological hypertrophy

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00338.2010

    mTOR overexpression in HL-1 cells. A : representative immunoblots of HA, mTOR, phospho-Akt, phospho-S6, total Akt, and total S6 in HL-1 cells transfected with mTOR. HL-1 cells were transfected with mock vector or mTOR using Lipofectamine 2000 as described
    Figure Legend Snippet: mTOR overexpression in HL-1 cells. A : representative immunoblots of HA, mTOR, phospho-Akt, phospho-S6, total Akt, and total S6 in HL-1 cells transfected with mTOR. HL-1 cells were transfected with mock vector or mTOR using Lipofectamine 2000 as described

    Techniques Used: Over Expression, Western Blot, Transfection, Plasmid Preparation

    10) Product Images from "Dysfunction of Platelet-derived Growth Factor Receptor α (PDGFRα) Represses the Production of Oligodendrocytes from Arylsulfatase A-deficient Multipotential Neural Precursor Cells *"

    Article Title: Dysfunction of Platelet-derived Growth Factor Receptor α (PDGFRα) Represses the Production of Oligodendrocytes from Arylsulfatase A-deficient Multipotential Neural Precursor Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.636498

    ASA-corrected ASA −/− NPs and recovery of PDGFRα . A , confocal immunocytochemical analysis of ASA (in green ) showed its association with Lamp1 + lysosomes (in red ) in ASA +/+ NPs and the absence of ASA expression in ASA−/− NPs. ASA−/− cells treated with 25% ASA conditioned medium exhibited association with lysosomes ( yellow ). B , ASA-corrected ASA−/− NPs showed a significant reduction in the ratio of long (C24:0 and C24:1) versus short (C16:0 and C18:0) fatty acid isoforms in treatments with 25% conditioned medium (ASACM) but not with 5 or 15% ASACM ( n = 3). *, p ≤ 0.05; **, p ≤ 0.01 by analysis of variance. C , immunoblotting analysis of ASA-corrected ASA −/− NPs showed a normalization of PDGFRα levels in NPs cell lysates. D , PDGFRα protein expression levels also recovered in DRMs and decreased in exosomes from enzymatically corrected ASA −/− NPs. E , AKT Ser 473 but not Thr 308 showed a significant decrease in phosphorylation in ASA −/− NPs. F , ERK1 phosphorylation at Thr 202 , ERK2 at Tyr 204 was similar in both cell types (data shown as the mean ± S.E.; n = 3). *, p
    Figure Legend Snippet: ASA-corrected ASA −/− NPs and recovery of PDGFRα . A , confocal immunocytochemical analysis of ASA (in green ) showed its association with Lamp1 + lysosomes (in red ) in ASA +/+ NPs and the absence of ASA expression in ASA−/− NPs. ASA−/− cells treated with 25% ASA conditioned medium exhibited association with lysosomes ( yellow ). B , ASA-corrected ASA−/− NPs showed a significant reduction in the ratio of long (C24:0 and C24:1) versus short (C16:0 and C18:0) fatty acid isoforms in treatments with 25% conditioned medium (ASACM) but not with 5 or 15% ASACM ( n = 3). *, p ≤ 0.05; **, p ≤ 0.01 by analysis of variance. C , immunoblotting analysis of ASA-corrected ASA −/− NPs showed a normalization of PDGFRα levels in NPs cell lysates. D , PDGFRα protein expression levels also recovered in DRMs and decreased in exosomes from enzymatically corrected ASA −/− NPs. E , AKT Ser 473 but not Thr 308 showed a significant decrease in phosphorylation in ASA −/− NPs. F , ERK1 phosphorylation at Thr 202 , ERK2 at Tyr 204 was similar in both cell types (data shown as the mean ± S.E.; n = 3). *, p

    Techniques Used: Expressing

    11) Product Images from "Involvement of toll-like receptor 4 in alveolar bone loss and glucose homeostasis in experimental periodontitis"

    Article Title: Involvement of toll-like receptor 4 in alveolar bone loss and glucose homeostasis in experimental periodontitis

    Journal: Journal of periodontal research

    doi: 10.1111/j.1600-0765.2010.01304.x

    (A) Akt phosphorylation in liver as determined by Western blot analysis. Upper panel: Western blot (representative of four independent experiments). Lower panel: pAkt/Akt ratio ( y -axis) determined from densitometric scanning of four independent western
    Figure Legend Snippet: (A) Akt phosphorylation in liver as determined by Western blot analysis. Upper panel: Western blot (representative of four independent experiments). Lower panel: pAkt/Akt ratio ( y -axis) determined from densitometric scanning of four independent western

    Techniques Used: Western Blot

    12) Product Images from "TMEM16A Induces MAPK and Contributes Directly to Tumorigenesis and Cancer Progression"

    Article Title: TMEM16A Induces MAPK and Contributes Directly to Tumorigenesis and Cancer Progression

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-12-0475-T

    The mitogenic phenotype of TMEM16A is associated with ERK1/2 activation. TMEM16A overexpression induced phospho-ERK1/2 and cyclin D1 (A). TMEM16A-siRNA led to decreased phospho-ERK1/2 and cyclin D1. Phospho-Akt levels were not consistently modulated by
    Figure Legend Snippet: The mitogenic phenotype of TMEM16A is associated with ERK1/2 activation. TMEM16A overexpression induced phospho-ERK1/2 and cyclin D1 (A). TMEM16A-siRNA led to decreased phospho-ERK1/2 and cyclin D1. Phospho-Akt levels were not consistently modulated by

    Techniques Used: Activation Assay, Over Expression

    13) Product Images from "Acquired resistance to dasatinib in lung cancer cell lines conferred by DDR2 gatekeeper mutation and NF1 loss"

    Article Title: Acquired resistance to dasatinib in lung cancer cell lines conferred by DDR2 gatekeeper mutation and NF1 loss

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-13-0817

    p-ERK levels are maintained in dasatinib resistant, but not parental, NCI-H2286 following dasatinib treatment. Immunoblots showing relative levels of the proteins ERK, Src, MEK, and AKT and phosphoproteins p-ERK (Thr202/Tyr204), p-Src (Tyr416), p-MEK
    Figure Legend Snippet: p-ERK levels are maintained in dasatinib resistant, but not parental, NCI-H2286 following dasatinib treatment. Immunoblots showing relative levels of the proteins ERK, Src, MEK, and AKT and phosphoproteins p-ERK (Thr202/Tyr204), p-Src (Tyr416), p-MEK

    Techniques Used: Western Blot

    14) Product Images from "MEF2D Deficiency in Neonatal Cardiomyocytes Triggers Cell Cycle Re-entry and Programmed Cell Death in Vitro *"

    Article Title: MEF2D Deficiency in Neonatal Cardiomyocytes Triggers Cell Cycle Re-entry and Programmed Cell Death in Vitro *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.666461

    PI3K/Akt inhibition rescues apoptotic cell death associated with MEF2D depletion. A , summary of apoptosis-associated genes dysregulated by 1.5-fold or more in MEF2D-depleted NRVMs. B , quantitative RT-PCR analysis of the activating E2Fs, E2f1 , E2f2 , and E2f3 , revealed that MEF2D deficiency results in their increased expression. C , two pro-apoptotic transcriptional targets of E2F, Apaf1 and Casp8 , are significantly up-regulated in MEF2D depleted NRVMs. Up-regulation of E2f1 , E2f2 , and E2f3 transcript levels ( D ) and the E2F pro-apoptotic targets Apaf1 and Casp8 ( E ) is abolished when PI3K/Akt activation is inhibited by the addition of GDC-0941 in MEF2D-depleted NRVMs. F , TUNEL assay of NRVMs treated with GDC-0941 demonstrates an abrogation of apoptosis observed in MEF2D-deficient NRVMs 5 days post-transduction. G , post-natal cardiomyocytes are terminally differentiated and are unable to re-enter the cell cycle. MEF2D depletion results in an up-regulation of Mcm3 , Mcm5 , Mcm6 , Pcna , Ccne1 , and Ccne2 and aberrant cell cycle re-entry. E2F transcription factors likely sense unprogrammed cell cycle activation and mediate cardiomyocyte apoptosis by transcriptionally activating pro-apoptotic genes Apaf1 and Casp8. Apaf1 , apoptotic peptidase-activating factor 1; Casp8 , caspase-8. The data are means ± S.E. ( error bars ). *, p
    Figure Legend Snippet: PI3K/Akt inhibition rescues apoptotic cell death associated with MEF2D depletion. A , summary of apoptosis-associated genes dysregulated by 1.5-fold or more in MEF2D-depleted NRVMs. B , quantitative RT-PCR analysis of the activating E2Fs, E2f1 , E2f2 , and E2f3 , revealed that MEF2D deficiency results in their increased expression. C , two pro-apoptotic transcriptional targets of E2F, Apaf1 and Casp8 , are significantly up-regulated in MEF2D depleted NRVMs. Up-regulation of E2f1 , E2f2 , and E2f3 transcript levels ( D ) and the E2F pro-apoptotic targets Apaf1 and Casp8 ( E ) is abolished when PI3K/Akt activation is inhibited by the addition of GDC-0941 in MEF2D-depleted NRVMs. F , TUNEL assay of NRVMs treated with GDC-0941 demonstrates an abrogation of apoptosis observed in MEF2D-deficient NRVMs 5 days post-transduction. G , post-natal cardiomyocytes are terminally differentiated and are unable to re-enter the cell cycle. MEF2D depletion results in an up-regulation of Mcm3 , Mcm5 , Mcm6 , Pcna , Ccne1 , and Ccne2 and aberrant cell cycle re-entry. E2F transcription factors likely sense unprogrammed cell cycle activation and mediate cardiomyocyte apoptosis by transcriptionally activating pro-apoptotic genes Apaf1 and Casp8. Apaf1 , apoptotic peptidase-activating factor 1; Casp8 , caspase-8. The data are means ± S.E. ( error bars ). *, p

    Techniques Used: Inhibition, Quantitative RT-PCR, Expressing, Activation Assay, TUNEL Assay, Transduction

    MEF2D overexpression in NRVMs prolongs survival. MEF2D overexpression for 5 d ( A ) and 7 d ( B ) resulted in increased cell viability of cultured cardiomyocytes. C , there was no change in caspase-3 activity 5 d after transduction in MEF2D-overexpressing cells. D , 7 d post-transduction, MEF2D-overexpressing NRVMs displayed a significant decrease in caspase-3 activity. E , Western blot analysis and quantification reveal a significant decrease in Akt phosphorylation at the activating residues threonine 308 and serine 473 in MEF2D-overexpressing cardiomyocytes. F , Western blot analysis and quantification revealed a significant increase in PTEN in MEF2D-overexpressing cardiomyocytes. OE , overexpression. The data are means ± S.E. *, p
    Figure Legend Snippet: MEF2D overexpression in NRVMs prolongs survival. MEF2D overexpression for 5 d ( A ) and 7 d ( B ) resulted in increased cell viability of cultured cardiomyocytes. C , there was no change in caspase-3 activity 5 d after transduction in MEF2D-overexpressing cells. D , 7 d post-transduction, MEF2D-overexpressing NRVMs displayed a significant decrease in caspase-3 activity. E , Western blot analysis and quantification reveal a significant decrease in Akt phosphorylation at the activating residues threonine 308 and serine 473 in MEF2D-overexpressing cardiomyocytes. F , Western blot analysis and quantification revealed a significant increase in PTEN in MEF2D-overexpressing cardiomyocytes. OE , overexpression. The data are means ± S.E. *, p

    Techniques Used: Over Expression, Cell Culture, Activity Assay, Transduction, Western Blot

    15) Product Images from "The protective effect of trimetazidine on myocardial ischemia/reperfusion injury through activating AMPK and ERK signaling pathway"

    Article Title: The protective effect of trimetazidine on myocardial ischemia/reperfusion injury through activating AMPK and ERK signaling pathway

    Journal: Metabolism: clinical and experimental

    doi: 10.1016/j.metabol.2015.10.022

    The levels of p-AMPK, p-ACC, p-ERK1/2 and p-AKT. The mice were subjected to 20 minutes of ischemia followed by 15 minutes of reperfusion. (A) Trimetazidine or vehicle (saline) was injected intravenously via the tail vein injection 5 minutes before reperfusion.
    Figure Legend Snippet: The levels of p-AMPK, p-ACC, p-ERK1/2 and p-AKT. The mice were subjected to 20 minutes of ischemia followed by 15 minutes of reperfusion. (A) Trimetazidine or vehicle (saline) was injected intravenously via the tail vein injection 5 minutes before reperfusion.

    Techniques Used: Mouse Assay, Injection

    16) Product Images from "SLUG is a Direct Transcriptional Repressor of PTEN Tumor Suppressor"

    Article Title: SLUG is a Direct Transcriptional Repressor of PTEN Tumor Suppressor

    Journal: The Prostate

    doi: 10.1002/pros.22974

    PTEN expression is regulated by endogenous SLUG in vivo ( A ) Western-blot analysis of AKT and PTEN expression in MEFs. Total protein was extracted from Slug+/+, Slug+/−, and Slug−/− MEFs (passage 1), and analyzed by Western-blot analysis using anti-pAKT, anti-PTEN, anti-AKT and anti-GAPDH (loading control) antibodies, respectively. ( B ) qPCR analysis of PTEN transcripts in MEFs. Total RNA was extracted from the indicated MEFs and synthesized into cDNAs. Using specific primers, qPCR was used to analyze PTEN transcripts. ** p
    Figure Legend Snippet: PTEN expression is regulated by endogenous SLUG in vivo ( A ) Western-blot analysis of AKT and PTEN expression in MEFs. Total protein was extracted from Slug+/+, Slug+/−, and Slug−/− MEFs (passage 1), and analyzed by Western-blot analysis using anti-pAKT, anti-PTEN, anti-AKT and anti-GAPDH (loading control) antibodies, respectively. ( B ) qPCR analysis of PTEN transcripts in MEFs. Total RNA was extracted from the indicated MEFs and synthesized into cDNAs. Using specific primers, qPCR was used to analyze PTEN transcripts. ** p

    Techniques Used: Expressing, In Vivo, Western Blot, Real-time Polymerase Chain Reaction, Synthesized

    17) Product Images from "Overexpression of AKIP1 predicts poor prognosis of patients with breast carcinoma and promotes cancer metastasis through Akt/GSK-3β/Snail pathway"

    Article Title: Overexpression of AKIP1 predicts poor prognosis of patients with breast carcinoma and promotes cancer metastasis through Akt/GSK-3β/Snail pathway

    Journal: American Journal of Translational Research

    doi:

    Down-regulation of AKIP1 inhibited Akt/GSK-3β/Snail signaling pathway. A. MCF-7 and SK-BR-3 cells were treated with AKIP1 si-RNA, and control si-RNA, following which cells were harvested for analysis of AKIP1, p-GSK-3β, GSK-3β,
    Figure Legend Snippet: Down-regulation of AKIP1 inhibited Akt/GSK-3β/Snail signaling pathway. A. MCF-7 and SK-BR-3 cells were treated with AKIP1 si-RNA, and control si-RNA, following which cells were harvested for analysis of AKIP1, p-GSK-3β, GSK-3β,

    Techniques Used:

    18) Product Images from "Alternatively spliced RAGEv1 inhibits tumorigenesis via suppression of JNK signaling"

    Article Title: Alternatively spliced RAGEv1 inhibits tumorigenesis via suppression of JNK signaling

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-10-0595

    RAGEv1 inhibits tumorigenesis through blocking a JNK-dependent signaling mechanism. ( A-C ) Cells were incubated with control (DMSO) or inhibitors for SAPK/JNK (SP600125), MEK1/2 (U0126), p38 (SB203580) and AKT (Triciribine) for 1h before stimulation with RAGE-ligand (s100B). RNA was then extracted and QPCR performed for APAF1, PDGFB and TNF, normalized to GAPDH levels. ( D ) JNK inhibition blocks RAGE-ligand induced tumor cell invasion. Matrigel invasion assays were performed with mock transfected cells, treated with control (DMSO) or SAPK/JNK (SP600125) and in response to vehicle or RAGE-ligand (s100B). Data are means ± s.e.m from three independent experiments. Significant differences (P ≤ 0.05) between groups are indicated by an asterisk.
    Figure Legend Snippet: RAGEv1 inhibits tumorigenesis through blocking a JNK-dependent signaling mechanism. ( A-C ) Cells were incubated with control (DMSO) or inhibitors for SAPK/JNK (SP600125), MEK1/2 (U0126), p38 (SB203580) and AKT (Triciribine) for 1h before stimulation with RAGE-ligand (s100B). RNA was then extracted and QPCR performed for APAF1, PDGFB and TNF, normalized to GAPDH levels. ( D ) JNK inhibition blocks RAGE-ligand induced tumor cell invasion. Matrigel invasion assays were performed with mock transfected cells, treated with control (DMSO) or SAPK/JNK (SP600125) and in response to vehicle or RAGE-ligand (s100B). Data are means ± s.e.m from three independent experiments. Significant differences (P ≤ 0.05) between groups are indicated by an asterisk.

    Techniques Used: Blocking Assay, Incubation, Real-time Polymerase Chain Reaction, Inhibition, Transfection

    RAGEv1 inhibits MAPK signaling ( A-D ) Cells (mock and RAGEv1) were stimulated with RAGE-ligand for the indicated times, lysed and subjected to western blot with antibodies for phospho-status and total of SAPK/JNK, MEK 1/2, p38 and AKT for the indicated times. Activity levels are expressed as a ratio of phospho/total levels. Data are means ± s.e.m from three independent experiments. Significant differences (P ≤ 0.05) between groups are indicated by an asterisk.
    Figure Legend Snippet: RAGEv1 inhibits MAPK signaling ( A-D ) Cells (mock and RAGEv1) were stimulated with RAGE-ligand for the indicated times, lysed and subjected to western blot with antibodies for phospho-status and total of SAPK/JNK, MEK 1/2, p38 and AKT for the indicated times. Activity levels are expressed as a ratio of phospho/total levels. Data are means ± s.e.m from three independent experiments. Significant differences (P ≤ 0.05) between groups are indicated by an asterisk.

    Techniques Used: Western Blot, Activity Assay

    19) Product Images from "A mouse model for Costello syndrome reveals an Ang II-mediated hypertensive condition"

    Article Title: A mouse model for Costello syndrome reveals an Ang II-mediated hypertensive condition

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI34385

    H-Ras signaling in heart and kidneys of H- Ras G12V mutant mice. Protein extracts (40 μg) obtained from ( A ) heart and kidneys of 2-month-old H- Ras –/– , H- Ras +/+ , H- Ras +/G12V , and H- Ras G12V/G12V mice and from ( B ) cardiomyocytes and fibroblasts isolated form neonatal hearts of H- Ras +/+ and H- Ras G12V/G12V animals were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with antibodies against H-Ras and the nonphosphorylated and phosphorylated forms of Erk1/2, Mek1, and Akt. GAPDH was used as loading control.
    Figure Legend Snippet: H-Ras signaling in heart and kidneys of H- Ras G12V mutant mice. Protein extracts (40 μg) obtained from ( A ) heart and kidneys of 2-month-old H- Ras –/– , H- Ras +/+ , H- Ras +/G12V , and H- Ras G12V/G12V mice and from ( B ) cardiomyocytes and fibroblasts isolated form neonatal hearts of H- Ras +/+ and H- Ras G12V/G12V animals were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with antibodies against H-Ras and the nonphosphorylated and phosphorylated forms of Erk1/2, Mek1, and Akt. GAPDH was used as loading control.

    Techniques Used: Mutagenesis, Mouse Assay, Isolation, SDS Page

    Functional characterization of the H-Ras signaling pathway in H- Ras G12V adult brain and E14.5 embryos. ( A ) Western blot analysis of the expression levels of wild-type H-Ras +/+ and mutant H-Ras G12V proteins. Protein extracts (200 μg) derived from colons of H- Ras +/+ , H- Ras +/G12V , H- Ras G12V/G12V , and H- Ras –/– mice as well as from brain (Br), heart (He), lung (Lu), kidney (Ki), and liver (Li) of H- Ras +/G12V animals were subjected to Western blot analysis. In the case of brain, only one-fourth of the sample was loaded. Migration of the wild-type H-Ras and mutant H-Ras G12V proteins is indicated by arrowheads. ( B ) Protein extracts (40 μg) from the indicated genotypes obtained from 2-month-old adult brains and E14.5 whole embryos were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with antibodies against H-Ras and the nonphosphorylated and phosphorylated forms of Erk1/2, Mek1, and Akt. Levels of active H-Ras protein bound to GTP (H-Ras–GTP) were determined by its ability to interact with the Ras binding domain of c-Raf. GAPDH was used as loading control.
    Figure Legend Snippet: Functional characterization of the H-Ras signaling pathway in H- Ras G12V adult brain and E14.5 embryos. ( A ) Western blot analysis of the expression levels of wild-type H-Ras +/+ and mutant H-Ras G12V proteins. Protein extracts (200 μg) derived from colons of H- Ras +/+ , H- Ras +/G12V , H- Ras G12V/G12V , and H- Ras –/– mice as well as from brain (Br), heart (He), lung (Lu), kidney (Ki), and liver (Li) of H- Ras +/G12V animals were subjected to Western blot analysis. In the case of brain, only one-fourth of the sample was loaded. Migration of the wild-type H-Ras and mutant H-Ras G12V proteins is indicated by arrowheads. ( B ) Protein extracts (40 μg) from the indicated genotypes obtained from 2-month-old adult brains and E14.5 whole embryos were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with antibodies against H-Ras and the nonphosphorylated and phosphorylated forms of Erk1/2, Mek1, and Akt. Levels of active H-Ras protein bound to GTP (H-Ras–GTP) were determined by its ability to interact with the Ras binding domain of c-Raf. GAPDH was used as loading control.

    Techniques Used: Functional Assay, Western Blot, Expressing, Mutagenesis, Derivative Assay, Mouse Assay, Migration, SDS Page, Binding Assay

    20) Product Images from "The effects of insulin on the inflammatory activity of BV2 microglia"

    Article Title: The effects of insulin on the inflammatory activity of BV2 microglia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201878

    Insulin administration increases AKT thr308 phosphorylation in BV2 cells. Insulin (0.36uM) was administered 1 hour after LPS to BV2 cells and AKT phosphorylation at thr308 or ser473 was assessed 2 hours later. A) Insulin alone significantly increased AKT thr308 phosphorylation. LPS also led to an increase in phosphorylation, but this was not significantly different than the control group. Insulin in addition to LPS led to no significant change in AKT phosphorylation from control or the LPS alone group. *p
    Figure Legend Snippet: Insulin administration increases AKT thr308 phosphorylation in BV2 cells. Insulin (0.36uM) was administered 1 hour after LPS to BV2 cells and AKT phosphorylation at thr308 or ser473 was assessed 2 hours later. A) Insulin alone significantly increased AKT thr308 phosphorylation. LPS also led to an increase in phosphorylation, but this was not significantly different than the control group. Insulin in addition to LPS led to no significant change in AKT phosphorylation from control or the LPS alone group. *p

    Techniques Used:

    21) Product Images from "Cholinesterase inhibitor rivastigmine enhances nerve growth factor-induced neurite outgrowth in PC12 cells via sigma-1 and sigma-2 receptors"

    Article Title: Cholinesterase inhibitor rivastigmine enhances nerve growth factor-induced neurite outgrowth in PC12 cells via sigma-1 and sigma-2 receptors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0209250

    Effect of Sig-2R antagonist and additional Sig-1R antagonist on Riv-mediated enhancement of NGF-induced neurite outgrowth and phosphorylation of Akt and ERK1/2 in PC12 cells. ( A ) PC12 cells were pre-incubated in the presence or absence of SM-21 (10 μM) for 4 h. ( B, C ) PC12 cells were pre-incubated in the presence or absence of SM-21 (10 μM) and NE-100 (10 μM) for 4 h. NGF and Riv were added, and cells were incubated for an additional 24 h before neurite outgrowth and western blotting assays. ( C ) Total protein lysates were collected and subjected to western blotting for the assessment of the phosphorylation status of Akt and ERK1/2. ( D , E ) The intensity of the p-Akt polypeptide band was normalized to that of the total Akt band (p-Akt/Akt) ( D ), while the intensity of the p-ERK1/2 polypeptide band was normalized to that of the total ERK1/2 band (p-ERK1/2/ERK1/2) ( E ). For each condition, values are reported as the mean ± SD for all PC12 cells included within five randomly chosen fields. ** p
    Figure Legend Snippet: Effect of Sig-2R antagonist and additional Sig-1R antagonist on Riv-mediated enhancement of NGF-induced neurite outgrowth and phosphorylation of Akt and ERK1/2 in PC12 cells. ( A ) PC12 cells were pre-incubated in the presence or absence of SM-21 (10 μM) for 4 h. ( B, C ) PC12 cells were pre-incubated in the presence or absence of SM-21 (10 μM) and NE-100 (10 μM) for 4 h. NGF and Riv were added, and cells were incubated for an additional 24 h before neurite outgrowth and western blotting assays. ( C ) Total protein lysates were collected and subjected to western blotting for the assessment of the phosphorylation status of Akt and ERK1/2. ( D , E ) The intensity of the p-Akt polypeptide band was normalized to that of the total Akt band (p-Akt/Akt) ( D ), while the intensity of the p-ERK1/2 polypeptide band was normalized to that of the total ERK1/2 band (p-ERK1/2/ERK1/2) ( E ). For each condition, values are reported as the mean ± SD for all PC12 cells included within five randomly chosen fields. ** p

    Techniques Used: Incubation, Western Blot

    Influence of Riv on NGF-induced phosphorylation of Akt and ERK1/2 in PC12 cells. PC12 cells underwent various durations of treatment (0, 10, 30, or 60 min) with NGF in the presence or absence of Riv (100 μM) ( A , C ) Total protein lysates were collected and subjected to western blotting for detection of phosphorylation status of Akt and ERK1/2. ( B , D ) The intensity of the p-Akt polypeptide band was normalized to that of the total Akt band (p-Akt/Akt) ( B ), while the intensity of the p-ERK1/2 polypeptide band was normalized to that of the total ERK1/2 band (p-ERK1/2/ERK1/2) ( D ). Results are presented as the mean ± SD of three independent experiments.
    Figure Legend Snippet: Influence of Riv on NGF-induced phosphorylation of Akt and ERK1/2 in PC12 cells. PC12 cells underwent various durations of treatment (0, 10, 30, or 60 min) with NGF in the presence or absence of Riv (100 μM) ( A , C ) Total protein lysates were collected and subjected to western blotting for detection of phosphorylation status of Akt and ERK1/2. ( B , D ) The intensity of the p-Akt polypeptide band was normalized to that of the total Akt band (p-Akt/Akt) ( B ), while the intensity of the p-ERK1/2 polypeptide band was normalized to that of the total ERK1/2 band (p-ERK1/2/ERK1/2) ( D ). Results are presented as the mean ± SD of three independent experiments.

    Techniques Used: Western Blot

    22) Product Images from "Pyrroline-5-carboxylate reductase 1 promotes cell proliferation via inhibiting apoptosis in human malignant melanoma"

    Article Title: Pyrroline-5-carboxylate reductase 1 promotes cell proliferation via inhibiting apoptosis in human malignant melanoma

    Journal: Cancer Management and Research

    doi: 10.2147/CMAR.S166711

    PYCR1 played a role in tumor progression of human MM through the stimulation of the AKT pathway. Notes: ( A ) The expression of AKT signaling pathway-related genes was analyzed by Western blot. ( B and C ) Statistical analysis of Western blot results was performed using ImageJ software. siPYCR1 inhibited AKT phosphorylation and P70 level, and promoted the expression of the apoptosis factors, Caspase3-p17 and Bax in A375 and M14 cell lines. ( D and E ) Rescue experiments were performed by activating the AKT pathway with IGF-1 in PYCR1 interference cells (siPYCR1+ IGF-1). CCK8 was used to detect cell proliferation in three cell groups. ( F ) The expression of AKT signaling pathway-related genes was analyzed by Western blot. ( G ) Statistical analysis of Western blot results was performed using ImageJ software. * P
    Figure Legend Snippet: PYCR1 played a role in tumor progression of human MM through the stimulation of the AKT pathway. Notes: ( A ) The expression of AKT signaling pathway-related genes was analyzed by Western blot. ( B and C ) Statistical analysis of Western blot results was performed using ImageJ software. siPYCR1 inhibited AKT phosphorylation and P70 level, and promoted the expression of the apoptosis factors, Caspase3-p17 and Bax in A375 and M14 cell lines. ( D and E ) Rescue experiments were performed by activating the AKT pathway with IGF-1 in PYCR1 interference cells (siPYCR1+ IGF-1). CCK8 was used to detect cell proliferation in three cell groups. ( F ) The expression of AKT signaling pathway-related genes was analyzed by Western blot. ( G ) Statistical analysis of Western blot results was performed using ImageJ software. * P

    Techniques Used: Expressing, Western Blot, Software

    23) Product Images from "The non-peptide thrombopoietin receptor agonist eltrombopag stimulates megakaryopoiesis in bone marrow cells from patients with relapsed multiple myeloma"

    Article Title: The non-peptide thrombopoietin receptor agonist eltrombopag stimulates megakaryopoiesis in bone marrow cells from patients with relapsed multiple myeloma

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-015-0136-2

    Eltrombopag induces Akt in human platelets and immature megakaryocytes. (A) Platelets from a healthy donor or (B) multiple myeloma-derived peripheral blood mobilized-CD34+ cells (MM#1 and MM#2) cultured for 8 days were stimulated with 100 ng/ml rhTPO or 10 μM eltrombopag for the indicated times, and immunoblotting was performed to detect the activation of STAT5 and Akt signaling pathways. Note that p-Akt bands appeared below non-specific bands in human platelets (A) and above non-specific bands in immature megakaryocytes (B) . Migration of molecular weight markers is indicated to the right of each blot.
    Figure Legend Snippet: Eltrombopag induces Akt in human platelets and immature megakaryocytes. (A) Platelets from a healthy donor or (B) multiple myeloma-derived peripheral blood mobilized-CD34+ cells (MM#1 and MM#2) cultured for 8 days were stimulated with 100 ng/ml rhTPO or 10 μM eltrombopag for the indicated times, and immunoblotting was performed to detect the activation of STAT5 and Akt signaling pathways. Note that p-Akt bands appeared below non-specific bands in human platelets (A) and above non-specific bands in immature megakaryocytes (B) . Migration of molecular weight markers is indicated to the right of each blot.

    Techniques Used: Derivative Assay, Cell Culture, Activation Assay, Migration, Molecular Weight

    24) Product Images from "Hypoxia Pretreatment of Bone Marrow Mesenchymal Stem Cells Facilitates Angiogenesis by Improving the Function of Endothelial Cells in Diabetic Rats with Lower Ischemia"

    Article Title: Hypoxia Pretreatment of Bone Marrow Mesenchymal Stem Cells Facilitates Angiogenesis by Improving the Function of Endothelial Cells in Diabetic Rats with Lower Ischemia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126715

    Hypoxia pretreatment of BM-MSCs modulates VEGF/AKT signaling in HUVECs. (A) The expression of VEGF-1α was suppressed with lentiviral-mediated siRNA-VEGF-1α (siVEGF). (B) CM generated from hypoxia pretreated BM-MSCs treated with siVEGF downregulated the expression of pAKT and VEGFR in HUVECs, as determined by western blot. (C) The capacity of HUVECs to form tubes was inhibited by CM from VEGF-1α-silenced BM-MSCs.
    Figure Legend Snippet: Hypoxia pretreatment of BM-MSCs modulates VEGF/AKT signaling in HUVECs. (A) The expression of VEGF-1α was suppressed with lentiviral-mediated siRNA-VEGF-1α (siVEGF). (B) CM generated from hypoxia pretreated BM-MSCs treated with siVEGF downregulated the expression of pAKT and VEGFR in HUVECs, as determined by western blot. (C) The capacity of HUVECs to form tubes was inhibited by CM from VEGF-1α-silenced BM-MSCs.

    Techniques Used: Expressing, Generated, Western Blot

    25) Product Images from "Iron Overload and Diabetes Risk: A Shift From Glucose to Fatty Acid Oxidation and Increased Hepatic Glucose Production in a Mouse Model of Hereditary Hemochromatosis"

    Article Title: Iron Overload and Diabetes Risk: A Shift From Glucose to Fatty Acid Oxidation and Increased Hepatic Glucose Production in a Mouse Model of Hereditary Hemochromatosis

    Journal: Diabetes

    doi: 10.2337/db10-0593

    Hepatic glucose metabolism in wild-type and Hfe −/− mice (129 strain) on normal chow. A : Hepatic glucose output (HGO) was determined by isotope dilution during euglycemic hyperinsulinemic clamps performed at submaximal (5 mU/kg/min) insulin infusion ( N = 4–6 mice per group). B : Hepatic levels of mRNAs for glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) were determined by quantitative RT-PCR in liver. Results ( N = 5–6/group) were normalized to cyclophilin A. C : Pyruvate tolerance testing of wild-type ( N = 6) and Hfe −/− ( N = 8) mice was performed by measuring blood glucose after injection of 2 mg pyruvate/g body weight. D : Phosphorylation of Akt and IRS2 were determined by Western blotting liver extracts from fasted wild-type and Hfe −/− mice on normal chow. E : Phosphorylation of Akt and IRS2 were normalized to total Akt and GAPDH, respectively, and quantitated by densitometry ( N = 4 each). F : Levels of pyruvate and glycogen were determined in liver tissue ( N = 4–6 per group). * P
    Figure Legend Snippet: Hepatic glucose metabolism in wild-type and Hfe −/− mice (129 strain) on normal chow. A : Hepatic glucose output (HGO) was determined by isotope dilution during euglycemic hyperinsulinemic clamps performed at submaximal (5 mU/kg/min) insulin infusion ( N = 4–6 mice per group). B : Hepatic levels of mRNAs for glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) were determined by quantitative RT-PCR in liver. Results ( N = 5–6/group) were normalized to cyclophilin A. C : Pyruvate tolerance testing of wild-type ( N = 6) and Hfe −/− ( N = 8) mice was performed by measuring blood glucose after injection of 2 mg pyruvate/g body weight. D : Phosphorylation of Akt and IRS2 were determined by Western blotting liver extracts from fasted wild-type and Hfe −/− mice on normal chow. E : Phosphorylation of Akt and IRS2 were normalized to total Akt and GAPDH, respectively, and quantitated by densitometry ( N = 4 each). F : Levels of pyruvate and glycogen were determined in liver tissue ( N = 4–6 per group). * P

    Techniques Used: Mouse Assay, Isotope Dilution, Quantitative RT-PCR, Injection, Western Blot

    26) Product Images from "An Osteoblast-Derived Proteinase Controls Tumor Cell Survival via TGF-beta Activation in the Bone Microenvironment"

    Article Title: An Osteoblast-Derived Proteinase Controls Tumor Cell Survival via TGF-beta Activation in the Bone Microenvironment

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029862

    Osteoblast-derived MMP-2 impacts TGFβ activation and tumor survival in the in vivo tumor-bone microenvironment. A , The levels of TGFβ in normalized tumor-bone lysates derived from WT and MMP-2 −/− tumor or sham injected tibias was assessed by ELISA. B , Representative immunoblots for phospho-SMAD (pSMAD2), total smad2 (SMAD2), phospho-AKT (pAKT), total AKT (AKT) and actin (loading control) in the tumor-bone lysates derived from WT and MMP-2 −/− mice. Densitometry on immunoblots generated from tumor bone lysates of at least 5 animals per group was used to generate graphs of the ratio of pSMAD2/SMAD2 and pAKT/AKT. Data are mean ± SD. *, p
    Figure Legend Snippet: Osteoblast-derived MMP-2 impacts TGFβ activation and tumor survival in the in vivo tumor-bone microenvironment. A , The levels of TGFβ in normalized tumor-bone lysates derived from WT and MMP-2 −/− tumor or sham injected tibias was assessed by ELISA. B , Representative immunoblots for phospho-SMAD (pSMAD2), total smad2 (SMAD2), phospho-AKT (pAKT), total AKT (AKT) and actin (loading control) in the tumor-bone lysates derived from WT and MMP-2 −/− mice. Densitometry on immunoblots generated from tumor bone lysates of at least 5 animals per group was used to generate graphs of the ratio of pSMAD2/SMAD2 and pAKT/AKT. Data are mean ± SD. *, p

    Techniques Used: Derivative Assay, Activation Assay, In Vivo, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Mouse Assay, Generated

    27) Product Images from "Intravenous injection of neural progenitor cells improved depression-like behavior after cerebral ischemia"

    Article Title: Intravenous injection of neural progenitor cells improved depression-like behavior after cerebral ischemia

    Journal: Translational Psychiatry

    doi: 10.1038/tp.2011.32

    Effect of neural progenitor cell (NPC) injection on the levels of brain-derived neurotrophic factor (BDNF), phosphorylated CREB, TrkB, phosphorylated ERK and phosphorylated AKT on Day 28 after the embolism. Samples obtained from sham-operated (sham), vehicle-injected ischemic (vehicle) and NPC-injected (NPC) ischemic rats (ME) were solubilized and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Bands corresponding to BDNF ( a ), phospho-CREB ( b ), and TrkB ( c ), phosphorylated ERK ( d ) and phosphorylated Akt ( e ) were scanned, and scanned bands were normalized by actin ( a and c ), total CREB ( b ), total ERK ( d ) or total AKT ( e ) on the same blot. All results are presented as mean percentages of non-operated naïve rats±s.e.m. (BDNF and phospho-CREB: n =9 rats per group; TrkB: n =5–6 rats per group; and phospho-ERK and -AKT: n =6 rats per group). * Statistically significant difference from sham-operated rats ( P
    Figure Legend Snippet: Effect of neural progenitor cell (NPC) injection on the levels of brain-derived neurotrophic factor (BDNF), phosphorylated CREB, TrkB, phosphorylated ERK and phosphorylated AKT on Day 28 after the embolism. Samples obtained from sham-operated (sham), vehicle-injected ischemic (vehicle) and NPC-injected (NPC) ischemic rats (ME) were solubilized and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Bands corresponding to BDNF ( a ), phospho-CREB ( b ), and TrkB ( c ), phosphorylated ERK ( d ) and phosphorylated Akt ( e ) were scanned, and scanned bands were normalized by actin ( a and c ), total CREB ( b ), total ERK ( d ) or total AKT ( e ) on the same blot. All results are presented as mean percentages of non-operated naïve rats±s.e.m. (BDNF and phospho-CREB: n =9 rats per group; TrkB: n =5–6 rats per group; and phospho-ERK and -AKT: n =6 rats per group). * Statistically significant difference from sham-operated rats ( P

    Techniques Used: Injection, Derivative Assay, Polyacrylamide Gel Electrophoresis, SDS Page

    28) Product Images from "MicroRNA-20b and ERK1/2 pathway independently regulate the expression of tissue factor in hematopoietic and trophoblastic differentiation of human embryonic stem cells"

    Article Title: MicroRNA-20b and ERK1/2 pathway independently regulate the expression of tissue factor in hematopoietic and trophoblastic differentiation of human embryonic stem cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt332

    Erk1/2 signaling pathway involved in regulating tissue factor expression in trophoblastic and hematopoietic differentiation of human embryonic stem cells. (A) Western blot analysis of phosphorylated Erk1/2 and Akt in various types of cells showing that Erk1/2 signaling pathway is active in granulocyte–macrophage (G-M) cells and trophoblasts. p-Erk1/2, phosphorylated Erk1/2; t-Erk1/2, total Erk1/2; pAkt, phosphorylated Akt; t-Akt, total Akt. (B),(C) Decreased (B) mRNA and (C) protein levels of tissue factor (TF) in G-M cells or trophoblasts treated with Erk1/2-specific inhibitor, U0126. Cells were treated with 10 μM U0126 or dimethylsulfoxide (control) for 4-6 or 7-9 days before harvest for quantitative real-time polymerase chain reaction for CDX2, PU.1, and TF mRNA levels. Data were reported as the mean ± standard error of the percentage of the mRNA levels of CDX2, PU.1, and TF in cells from the control group. * P
    Figure Legend Snippet: Erk1/2 signaling pathway involved in regulating tissue factor expression in trophoblastic and hematopoietic differentiation of human embryonic stem cells. (A) Western blot analysis of phosphorylated Erk1/2 and Akt in various types of cells showing that Erk1/2 signaling pathway is active in granulocyte–macrophage (G-M) cells and trophoblasts. p-Erk1/2, phosphorylated Erk1/2; t-Erk1/2, total Erk1/2; pAkt, phosphorylated Akt; t-Akt, total Akt. (B),(C) Decreased (B) mRNA and (C) protein levels of tissue factor (TF) in G-M cells or trophoblasts treated with Erk1/2-specific inhibitor, U0126. Cells were treated with 10 μM U0126 or dimethylsulfoxide (control) for 4-6 or 7-9 days before harvest for quantitative real-time polymerase chain reaction for CDX2, PU.1, and TF mRNA levels. Data were reported as the mean ± standard error of the percentage of the mRNA levels of CDX2, PU.1, and TF in cells from the control group. * P

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction

    29) Product Images from "Vascular endothelial cells facilitated HCC invasion and metastasis through the Akt and NF-?B pathways induced by paracrine cytokines"

    Article Title: Vascular endothelial cells facilitated HCC invasion and metastasis through the Akt and NF-?B pathways induced by paracrine cytokines

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-32-51

    Effects of CM on PI3K/Akt and ERK pathway activation in HCC cells. (A) Expression of p-Akt and p-ERK in MHCC97H cells under CM or EBM stimulation were detected by Western blot. (B) Expression of p-Akt and p-ERK in subcutaneous tumors derived from a mixture of MHCC97H cells and HUVECs were analyzed by immunohistochemistry.
    Figure Legend Snippet: Effects of CM on PI3K/Akt and ERK pathway activation in HCC cells. (A) Expression of p-Akt and p-ERK in MHCC97H cells under CM or EBM stimulation were detected by Western blot. (B) Expression of p-Akt and p-ERK in subcutaneous tumors derived from a mixture of MHCC97H cells and HUVECs were analyzed by immunohistochemistry.

    Techniques Used: Activation Assay, Expressing, Western Blot, Derivative Assay, Immunohistochemistry

    Effects of CCL2, IL-8, and CXCL16 on the activation of the Akt, ERK, and NF-κB pathways in HCC cells. The levels of phosphorylated Akt and ERK in MHCC97H (A) Cells after exposure to CCL2, IL-8, or CXCL16 at different concentrations. (B) Activation of NF-κB in MHCC97H cells were measured using a specific TransAM NF-κB p65 kit under CCL2, IL-8, or CXCL16 stimulation (* P
    Figure Legend Snippet: Effects of CCL2, IL-8, and CXCL16 on the activation of the Akt, ERK, and NF-κB pathways in HCC cells. The levels of phosphorylated Akt and ERK in MHCC97H (A) Cells after exposure to CCL2, IL-8, or CXCL16 at different concentrations. (B) Activation of NF-κB in MHCC97H cells were measured using a specific TransAM NF-κB p65 kit under CCL2, IL-8, or CXCL16 stimulation (* P

    Techniques Used: Activation Assay

    30) Product Images from "Syntenin increases the invasiveness of small cell lung cancer cells by activating p38, AKT, focal adhesion kinase and SP1"

    Article Title: Syntenin increases the invasiveness of small cell lung cancer cells by activating p38, AKT, focal adhesion kinase and SP1

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/emm.2014.1

    Activation of p38, AKT and focal adhesion kinase by forced expression of syntenin in small cell lung cancer cells. ( a ) Western blot analysis revealed enhanced phosphorylation of p38 MAPK and AKT after syntenin overexpression in NCI-H187 cells, compared with empty-vector transfection. In contrast, phosphorylated AKT and p38 MAPK were shown to be decreased by syntenin inhibition in NCI-H69 cells. Activation of focal adhesion kinase (FAK) by syntenin overexpression and suppression of FAK by syntenin inhibition was also observed without stimulation of any extracellular matrix proteins. ( b ) Western blot analysis after administration of SB203580 (10 μ M ) revealed the inhibition of p38 activation in syntenin-transfected cells compared with the p38 level in empty vector-transfected cells, although the basal level itself was upregulated, due to the role of SB203580 as not only an inhibitor but also as a partial agonist (left). In RT–PCR analysis, the inhibition of p38 MAPK induced the inhibition of both MMP2 and MT1-MMP mRNA (right). ( c ) The administration of LY294002 (1 μ M ) suppressed AKT activation in syntenin-transfected cells (left), which led to the downregulation of MMP2 and MT1-MMP, despite syntenin overexpression (right). ( d ) The activation of FAK was inhibited by administration of PF-573228 (1 μ M ) (left), which antagonized the syntenin-mediated upregulation of MMP2 and MT1-MMP (right). ( e ) When FAK, P38 and PI3K/AKT were inhibited by pharmacological inhibitors in NCI-H187 cells, the number of invaded cells in the Matrigel invasion assay did not increase in syntenin-transfected cells. ( f ) In NCI-H69 cells, the number of invaded cells decreased after inhibition of p38 and PI3K/AKT. The invasiveness of NCI-H69 cells was suppressed by syntenin inhibition. MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MT1, membrane type 1.
    Figure Legend Snippet: Activation of p38, AKT and focal adhesion kinase by forced expression of syntenin in small cell lung cancer cells. ( a ) Western blot analysis revealed enhanced phosphorylation of p38 MAPK and AKT after syntenin overexpression in NCI-H187 cells, compared with empty-vector transfection. In contrast, phosphorylated AKT and p38 MAPK were shown to be decreased by syntenin inhibition in NCI-H69 cells. Activation of focal adhesion kinase (FAK) by syntenin overexpression and suppression of FAK by syntenin inhibition was also observed without stimulation of any extracellular matrix proteins. ( b ) Western blot analysis after administration of SB203580 (10 μ M ) revealed the inhibition of p38 activation in syntenin-transfected cells compared with the p38 level in empty vector-transfected cells, although the basal level itself was upregulated, due to the role of SB203580 as not only an inhibitor but also as a partial agonist (left). In RT–PCR analysis, the inhibition of p38 MAPK induced the inhibition of both MMP2 and MT1-MMP mRNA (right). ( c ) The administration of LY294002 (1 μ M ) suppressed AKT activation in syntenin-transfected cells (left), which led to the downregulation of MMP2 and MT1-MMP, despite syntenin overexpression (right). ( d ) The activation of FAK was inhibited by administration of PF-573228 (1 μ M ) (left), which antagonized the syntenin-mediated upregulation of MMP2 and MT1-MMP (right). ( e ) When FAK, P38 and PI3K/AKT were inhibited by pharmacological inhibitors in NCI-H187 cells, the number of invaded cells in the Matrigel invasion assay did not increase in syntenin-transfected cells. ( f ) In NCI-H69 cells, the number of invaded cells decreased after inhibition of p38 and PI3K/AKT. The invasiveness of NCI-H69 cells was suppressed by syntenin inhibition. MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MT1, membrane type 1.

    Techniques Used: Activation Assay, Expressing, Western Blot, Over Expression, Plasmid Preparation, Transfection, Inhibition, Reverse Transcription Polymerase Chain Reaction, Invasion Assay

    Schematic model of signaling pathways activated by syntenin activation in small cell lung cancer cells. Without extracellular matrix interactions, focal adhesion kinase (FAK) is phosphorylated by syntenin activation. The activated signal is then transduced via p38 MAPK and AKT phosphorylation. Through action of the gene-regulating element SP1, signal transduction progressed from the cytoplasm to nucleus. Finally, expression of MT1-MMP and MMP2 was induced, and these products then enhanced cell motility, invasion and tumor growth. MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MT1, membrane type 1.
    Figure Legend Snippet: Schematic model of signaling pathways activated by syntenin activation in small cell lung cancer cells. Without extracellular matrix interactions, focal adhesion kinase (FAK) is phosphorylated by syntenin activation. The activated signal is then transduced via p38 MAPK and AKT phosphorylation. Through action of the gene-regulating element SP1, signal transduction progressed from the cytoplasm to nucleus. Finally, expression of MT1-MMP and MMP2 was induced, and these products then enhanced cell motility, invasion and tumor growth. MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MT1, membrane type 1.

    Techniques Used: Activation Assay, Transduction, Expressing

    31) Product Images from "Scavenger receptor class B type I regulates cellular cholesterol metabolism and cell signaling associated with breast cancer development"

    Article Title: Scavenger receptor class B type I regulates cellular cholesterol metabolism and cell signaling associated with breast cancer development

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr3483

    Inhibition of PI3K, not MEK1/2, prevents proliferation of MDA-MB-231 cells. (A) LY294002 and U0126 effectively inhibit Akt and Erk1/2 activation in MDA-MB-231 cells. Serum-starved shCTL and shSRBI MDA-MB-231 cells were incubated with or without the inhibitors LY294002 (15 μ M ) or U0126 (10 μ M ) for 2 hours. Medium containing 10% FBS was added for 30 minutes, cells were lysed, and whole-cell lysates were analyzed by Western blot for the indicated proteins. GAPDH was used as a loading control. (B) PI3K inhibition reduces cellular proliferation of shCTL MDA-MB-231 cells. shCTL and shSRBI MDA-MB-231 cells were incubated with culture media containing either LY294002 (15 μ M ) or U0126 (10 μ M ) and 3 H-thymidine for 6 hours, at which time, the assay was stopped, and lysates were collected. Columns represent the mean [ 3 H]thymidine incorporation (cpm/μg protein); bars represent ± SD. Results obtained from vehicle-treated (CTL) shCTL MDA-MB-231 cells are significantly different from those obtained with shSRBI MDA-MB-231 cells (** P
    Figure Legend Snippet: Inhibition of PI3K, not MEK1/2, prevents proliferation of MDA-MB-231 cells. (A) LY294002 and U0126 effectively inhibit Akt and Erk1/2 activation in MDA-MB-231 cells. Serum-starved shCTL and shSRBI MDA-MB-231 cells were incubated with or without the inhibitors LY294002 (15 μ M ) or U0126 (10 μ M ) for 2 hours. Medium containing 10% FBS was added for 30 minutes, cells were lysed, and whole-cell lysates were analyzed by Western blot for the indicated proteins. GAPDH was used as a loading control. (B) PI3K inhibition reduces cellular proliferation of shCTL MDA-MB-231 cells. shCTL and shSRBI MDA-MB-231 cells were incubated with culture media containing either LY294002 (15 μ M ) or U0126 (10 μ M ) and 3 H-thymidine for 6 hours, at which time, the assay was stopped, and lysates were collected. Columns represent the mean [ 3 H]thymidine incorporation (cpm/μg protein); bars represent ± SD. Results obtained from vehicle-treated (CTL) shCTL MDA-MB-231 cells are significantly different from those obtained with shSRBI MDA-MB-231 cells (** P

    Techniques Used: Inhibition, Multiple Displacement Amplification, Activation Assay, Incubation, Western Blot, CTL Assay

    32) Product Images from "AP-2?: a regulator of EGF receptor signaling and proliferation in skin epidermis"

    Article Title: AP-2?: a regulator of EGF receptor signaling and proliferation in skin epidermis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200510002

    In vitro and in vivo superactivation of the PI3K–Akt pathway in an EGF-dependent and AP-2α null–dependent fashion. (A) Primary WT and KO keratinocytes were cultured ± the EGFR-specific protein kinase inhibitor AG1478 without the addition of fibroblast feeder cells after the original plating. Growth curves represents three independent experiments performed in duplicate (see Separation of epidermis…transfections). Error bars represent SD for these experiments. (B) Keratinocytes were serum-starved for 24 h and at t = 0, 50 ng/ml EGF was added. At the times indicated, cell extracts were prepared, proteins were resolved by SDS-PAGE, and immunoblot analyses were conducted with the antibodies indicated at right. The antibodies against the active forms of Erk1/2 and Akt (p-Erk and p-Akt, respectively) are phosphospecific antibodies and do not recognize the inactive states of the kinases. β-tubulin antibodies are used as a loading control, along with ponseau red staining of the blots (not depicted). Note EGF-dependent superactivation of Akt and Erk1/2 in AP-2α –null keratinocytes. (C) Same experiment as in B, except 10× increments of EGF concentrations (0–50 ng/ml) were used, and extracts were harvested at t = 2 min. (D) Same experiment as in B, but insulin growth factor 1 (IGF1) was added at 50 ng/ml. Note that in contrast to EGF, IGF1 did not generate enhanced activated Akt in KO relative to WT cells. (E) Same experiment as in B, but the PI3K-specific inhibitor LY294002 was added at 20 μM. Note that the activation of Akt, but not Erk1/2, is dependent on PI3K activation. (F) Antiphospho-Akt staining of frozen sections (8 μm) from (top) lesional KO and WT thoracic skins and (bottom) TPA-treated KO and WT back skins. Note the superactivation of Akt in KO skin regions that are lesional and that correlate with elevated EGFR ligand expression. De, dermis; epi, epidermis; hf, hair follicle. Dotted lines denote dermo–epidermal borders. Bars, 20 μm.
    Figure Legend Snippet: In vitro and in vivo superactivation of the PI3K–Akt pathway in an EGF-dependent and AP-2α null–dependent fashion. (A) Primary WT and KO keratinocytes were cultured ± the EGFR-specific protein kinase inhibitor AG1478 without the addition of fibroblast feeder cells after the original plating. Growth curves represents three independent experiments performed in duplicate (see Separation of epidermis…transfections). Error bars represent SD for these experiments. (B) Keratinocytes were serum-starved for 24 h and at t = 0, 50 ng/ml EGF was added. At the times indicated, cell extracts were prepared, proteins were resolved by SDS-PAGE, and immunoblot analyses were conducted with the antibodies indicated at right. The antibodies against the active forms of Erk1/2 and Akt (p-Erk and p-Akt, respectively) are phosphospecific antibodies and do not recognize the inactive states of the kinases. β-tubulin antibodies are used as a loading control, along with ponseau red staining of the blots (not depicted). Note EGF-dependent superactivation of Akt and Erk1/2 in AP-2α –null keratinocytes. (C) Same experiment as in B, except 10× increments of EGF concentrations (0–50 ng/ml) were used, and extracts were harvested at t = 2 min. (D) Same experiment as in B, but insulin growth factor 1 (IGF1) was added at 50 ng/ml. Note that in contrast to EGF, IGF1 did not generate enhanced activated Akt in KO relative to WT cells. (E) Same experiment as in B, but the PI3K-specific inhibitor LY294002 was added at 20 μM. Note that the activation of Akt, but not Erk1/2, is dependent on PI3K activation. (F) Antiphospho-Akt staining of frozen sections (8 μm) from (top) lesional KO and WT thoracic skins and (bottom) TPA-treated KO and WT back skins. Note the superactivation of Akt in KO skin regions that are lesional and that correlate with elevated EGFR ligand expression. De, dermis; epi, epidermis; hf, hair follicle. Dotted lines denote dermo–epidermal borders. Bars, 20 μm.

    Techniques Used: In Vitro, In Vivo, Cell Culture, SDS Page, Staining, Activation Assay, Expressing

    33) Product Images from "Reciprocal regulation of IL-23 and IL-12 following co-activation of Dectin-1 and TLR signaling pathways"

    Article Title: Reciprocal regulation of IL-23 and IL-12 following co-activation of Dectin-1 and TLR signaling pathways

    Journal: European Journal of Immunology

    doi: 10.1002/eji.200838543

    Down-regulation of IL-12 is dependent on signaling through Dectin-1. (A) Phosphorylation of Syk and Akt after stimulation of Balb/C BMDC with 10 μg/mL soluble β-glucan and 1 μg/mL Pam 3 CSK 4 , as indicated. Production of IL-10 (B) and IL-12p70 (C) from Balb/C Syk −/− or WT thioglycollate-elicited macrophages pretreated with vehicle, 20 μM U0126 or 20 μM Ly294002, and stimulated with 10 μg/mL β-glucan and 10 ng/mL Pam 3 CSK 4 , as indicated. (D) Production of IL-12p70 from Balb/C WT or 129Sv Dectin-1 −/− BMDC pretreated with vehicle, 20 μM U0126 or 20 μM Ly294002, and stimulated with 10 μg/mL β-glucan and 100 ng/mL Pam 3 CSK 4 , as indicated. (E) Production of IL-12p70 from Balb/C WT or IL-10 −/− BMDC stimulated with 10 μg/mL β-glucan and 100 ng/mL Pam 3 CSK 4 , as indicated. Data shown are mean±SD and are representative of two independent experiments.
    Figure Legend Snippet: Down-regulation of IL-12 is dependent on signaling through Dectin-1. (A) Phosphorylation of Syk and Akt after stimulation of Balb/C BMDC with 10 μg/mL soluble β-glucan and 1 μg/mL Pam 3 CSK 4 , as indicated. Production of IL-10 (B) and IL-12p70 (C) from Balb/C Syk −/− or WT thioglycollate-elicited macrophages pretreated with vehicle, 20 μM U0126 or 20 μM Ly294002, and stimulated with 10 μg/mL β-glucan and 10 ng/mL Pam 3 CSK 4 , as indicated. (D) Production of IL-12p70 from Balb/C WT or 129Sv Dectin-1 −/− BMDC pretreated with vehicle, 20 μM U0126 or 20 μM Ly294002, and stimulated with 10 μg/mL β-glucan and 100 ng/mL Pam 3 CSK 4 , as indicated. (E) Production of IL-12p70 from Balb/C WT or IL-10 −/− BMDC stimulated with 10 μg/mL β-glucan and 100 ng/mL Pam 3 CSK 4 , as indicated. Data shown are mean±SD and are representative of two independent experiments.

    Techniques Used:

    34) Product Images from "Akt regulates the expression of MafK, synaptotagmin I, and syntenin-1, which play roles in neuronal function"

    Article Title: Akt regulates the expression of MafK, synaptotagmin I, and syntenin-1, which play roles in neuronal function

    Journal: Journal of Biomedical Science

    doi: 10.1186/1423-0127-17-18

    Analyses of MafK , SytI , and Syn-1 mRNA levels in parental, WT-Akt-expressing, and DN-Akt-expressing PC12 cells . (A) Quantitative RT-PCR. mRNA level of the gene in each group of cells was normalized to the level of GAPDH mRNA and the transcript level of each gene in PC12 (WT-Akt) and PC12 (DN-Akt) cells is presented as a fold change from that of the PC12 (parental) cells. Parental, PC12 (parental) cells. * P
    Figure Legend Snippet: Analyses of MafK , SytI , and Syn-1 mRNA levels in parental, WT-Akt-expressing, and DN-Akt-expressing PC12 cells . (A) Quantitative RT-PCR. mRNA level of the gene in each group of cells was normalized to the level of GAPDH mRNA and the transcript level of each gene in PC12 (WT-Akt) and PC12 (DN-Akt) cells is presented as a fold change from that of the PC12 (parental) cells. Parental, PC12 (parental) cells. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of the Akt inhibitor AKTi-1/2 on mRNA levels for the MafK , SytI , and Syn-1 genes . (A) Western blot analysis of pAkt(Ser-473) protein level in PC12 (WT-Akt) cells treated with various concentrations of AKTi-1/2 prior to 10 min treatment with NGF. (B) The percentage of neuritogenic PC12 (WT-Akt) cells treated with or without AKTi-1/2. Averages and standard deviations are derived from more than six fields of view. * P
    Figure Legend Snippet: Effect of the Akt inhibitor AKTi-1/2 on mRNA levels for the MafK , SytI , and Syn-1 genes . (A) Western blot analysis of pAkt(Ser-473) protein level in PC12 (WT-Akt) cells treated with various concentrations of AKTi-1/2 prior to 10 min treatment with NGF. (B) The percentage of neuritogenic PC12 (WT-Akt) cells treated with or without AKTi-1/2. Averages and standard deviations are derived from more than six fields of view. * P

    Techniques Used: Western Blot, Derivative Assay

    35) Product Images from "EGCG reverses human neutrophil elastase-induced migration in A549 cells by directly binding to HNE and by regulating α1-AT"

    Article Title: EGCG reverses human neutrophil elastase-induced migration in A549 cells by directly binding to HNE and by regulating α1-AT

    Journal: Scientific Reports

    doi: 10.1038/srep11494

    EGCG regulates the neutrophil elastase-induced migration of A549 cells. EGCG inhibits the activity of neutrophil elastase by directly binding to neutrophil elastase. EGCG enhances the expression of AAT by up-regulating the Akt/PI3K signaling pathway.
    Figure Legend Snippet: EGCG regulates the neutrophil elastase-induced migration of A549 cells. EGCG inhibits the activity of neutrophil elastase by directly binding to neutrophil elastase. EGCG enhances the expression of AAT by up-regulating the Akt/PI3K signaling pathway.

    Techniques Used: Migration, Activity Assay, Binding Assay, Expressing

    EGCG attenuates the neutrophil elastase-induced cell migration via the PI3K/Akt pathway. ( A ) Western blot for AAT, IRS-1, pAkt (phosphorylated at Thy308 or Ser473), Akt, p-PI3K, PI3K and GAPDH in A549 cells treated with EGCG or sivelestat sodium with or without HNE. ( B - F ) The statistical data are presented as histograms of the Western blot results for AAT (B), IRS-1 ( C ), the pAkt (Thy308)/Akt ratio ( D ), the pAkt (Ser473)/Akt ratio ( E ) and the pPI3K/PI3K ratio ( F ) in A549 cells treated with neutrophil elastase, EGCG and sivelestat sodium. The data are presented as the means ± SEM; n = 3. **P
    Figure Legend Snippet: EGCG attenuates the neutrophil elastase-induced cell migration via the PI3K/Akt pathway. ( A ) Western blot for AAT, IRS-1, pAkt (phosphorylated at Thy308 or Ser473), Akt, p-PI3K, PI3K and GAPDH in A549 cells treated with EGCG or sivelestat sodium with or without HNE. ( B - F ) The statistical data are presented as histograms of the Western blot results for AAT (B), IRS-1 ( C ), the pAkt (Thy308)/Akt ratio ( D ), the pAkt (Ser473)/Akt ratio ( E ) and the pPI3K/PI3K ratio ( F ) in A549 cells treated with neutrophil elastase, EGCG and sivelestat sodium. The data are presented as the means ± SEM; n = 3. **P

    Techniques Used: Migration, Western Blot

    36) Product Images from "Protective role of 5-azacytidine on myocardial infarction is associated with modulation of macrophage phenotype and inhibition of fibrosis"

    Article Title: Protective role of 5-azacytidine on myocardial infarction is associated with modulation of macrophage phenotype and inhibition of fibrosis

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12248

    Effect of 5AZ on angiotensin II (AngII)-stimulated cardiac fibroblasts. (A) The cell number of cardiac fibroblasts was significantly increased after treatment with AngII for 5 days, and 5AZ blocked AngII-induced proliferation. (B) Increased mRNA of connective tissue growth factor (CTGF) and collagen type I (ColI), fibrosis-mediators, was reduced after 5AZ treatment. (C) AngII-induced expression of phosphorylated extracellular signal-regulated kinase, phosphorylated Akt and bcl-2 was reduced after 5AZ treatment. In bar graphs; (1) control; (2) AngII 0.1 μM for 1 day; (3) AngII 0.1 μM + 5AZ for 1 day; (4) AngII 1 μM for 1 day; (5) AngII 1 μM + 5AZ for 1 day; (6) AngII 0.1 μM for 5 days; (7) AngII 0.1 μM + 5AZ for 5 days; (8) AngII 1 μM for 5 days; (9) AngII 1 μM + 5AZ for 5 days.
    Figure Legend Snippet: Effect of 5AZ on angiotensin II (AngII)-stimulated cardiac fibroblasts. (A) The cell number of cardiac fibroblasts was significantly increased after treatment with AngII for 5 days, and 5AZ blocked AngII-induced proliferation. (B) Increased mRNA of connective tissue growth factor (CTGF) and collagen type I (ColI), fibrosis-mediators, was reduced after 5AZ treatment. (C) AngII-induced expression of phosphorylated extracellular signal-regulated kinase, phosphorylated Akt and bcl-2 was reduced after 5AZ treatment. In bar graphs; (1) control; (2) AngII 0.1 μM for 1 day; (3) AngII 0.1 μM + 5AZ for 1 day; (4) AngII 1 μM for 1 day; (5) AngII 1 μM + 5AZ for 1 day; (6) AngII 0.1 μM for 5 days; (7) AngII 0.1 μM + 5AZ for 5 days; (8) AngII 1 μM for 5 days; (9) AngII 1 μM + 5AZ for 5 days.

    Techniques Used: Expressing

    37) Product Images from "Non-canonical antagonism of PI3K by the kinase Itpkb delays thymocyte β-selection and renders it Notch-dependent"

    Article Title: Non-canonical antagonism of PI3K by the kinase Itpkb delays thymocyte β-selection and renders it Notch-dependent

    Journal: eLife

    doi: 10.7554/eLife.10786

    Increased pre-TCR signaling via PI3K/Akt/mTOR in Itpkb -/- DN3 cells. ( A,B ) We analyzed ( A ) cellular content of T 308 -phosphorylated active Akt (pAkt T308 ), S 2481 -phosphorylated mTOR (pmTOR S2481 ), S 235 /S 236 -phosphorylated ribosomal protein S6 (pS6 S235/S236 ), Glut1 protein, T 202 /Y 204 -phosphorylated Erk (pErk T202/Y204 ) and Akt protein, and ( B ) cell size via side/forward-scatter analysis (SSC-A/FSC-A) in the indicated thymocyte populations of Itpkb +/+ (black histograms) or Itpkb -/- (gray histograms) mice by FACS. Thin open histograms, Itpkb +/+ isotype or second antibody stained negative controls. Bold open histograms, Calyculin A-treated positive controls. Arrowheads show gate positions. In ( B ), numbers indicate % cells per large cell gate. Representative of at least 2 (pS6 S235/S236 ), 3 (total Akt) or 8 (else) independent experiments. DOI: http://dx.doi.org/10.7554/eLife.10786.011
    Figure Legend Snippet: Increased pre-TCR signaling via PI3K/Akt/mTOR in Itpkb -/- DN3 cells. ( A,B ) We analyzed ( A ) cellular content of T 308 -phosphorylated active Akt (pAkt T308 ), S 2481 -phosphorylated mTOR (pmTOR S2481 ), S 235 /S 236 -phosphorylated ribosomal protein S6 (pS6 S235/S236 ), Glut1 protein, T 202 /Y 204 -phosphorylated Erk (pErk T202/Y204 ) and Akt protein, and ( B ) cell size via side/forward-scatter analysis (SSC-A/FSC-A) in the indicated thymocyte populations of Itpkb +/+ (black histograms) or Itpkb -/- (gray histograms) mice by FACS. Thin open histograms, Itpkb +/+ isotype or second antibody stained negative controls. Bold open histograms, Calyculin A-treated positive controls. Arrowheads show gate positions. In ( B ), numbers indicate % cells per large cell gate. Representative of at least 2 (pS6 S235/S236 ), 3 (total Akt) or 8 (else) independent experiments. DOI: http://dx.doi.org/10.7554/eLife.10786.011

    Techniques Used: Mouse Assay, FACS, Staining

    38) Product Images from "Beyond proliferation: KLF5 promotes angiogenesis of bladder cancer through directly regulating VEGFA transcription"

    Article Title: Beyond proliferation: KLF5 promotes angiogenesis of bladder cancer through directly regulating VEGFA transcription

    Journal: Oncotarget

    doi:

    Therapeutic potential of targeting KLF5 in bladder cancer cells A. Serum-starved 5637 and WH cells were pre-treated with inhibitors of EGFR (AG1478, 5 μM), MEK1/2 (U0126, 5 μM) or PI3K (LY294002, 5 μM) before EGF (50 ng/ml) treatment for 12 hours. Then KLF5, VEGFA expression and phosphorylation status of Erk1/2 and Akt were evaluated by western blot. B. Impact of a KLF5 specific inhibitor (CID5951923) on the KLF5 and VEGFA protein expression as measured by western blot in 5637 and WH cells. C. Schematic model of PMA/LPA/RTKs-KLF5-VEGFA signaling and novel therapeutic targets in bladder cancer.
    Figure Legend Snippet: Therapeutic potential of targeting KLF5 in bladder cancer cells A. Serum-starved 5637 and WH cells were pre-treated with inhibitors of EGFR (AG1478, 5 μM), MEK1/2 (U0126, 5 μM) or PI3K (LY294002, 5 μM) before EGF (50 ng/ml) treatment for 12 hours. Then KLF5, VEGFA expression and phosphorylation status of Erk1/2 and Akt were evaluated by western blot. B. Impact of a KLF5 specific inhibitor (CID5951923) on the KLF5 and VEGFA protein expression as measured by western blot in 5637 and WH cells. C. Schematic model of PMA/LPA/RTKs-KLF5-VEGFA signaling and novel therapeutic targets in bladder cancer.

    Techniques Used: Expressing, Western Blot

    VEGFA mediates the angiogenic roles of KLF5 in bladder cancer A. VEGFA neutralized antibody (MAB293) was added into CMs from 5637/shNC cells, while recombinant human VEGF 165 was added into CMs from 5637/shKLF5 cells. These CMs containing different levels of VEGFA were used to recruit HUVECs. B. HUVECs were serum-starved overnight, stimulated with SFM or CMs for 10 minutes before proteins harvest immediately. Phosphorylation of VEGFR2, Erk1/2 and Akt were evaluated by western blot. These data were representative of three independent experiments; * p
    Figure Legend Snippet: VEGFA mediates the angiogenic roles of KLF5 in bladder cancer A. VEGFA neutralized antibody (MAB293) was added into CMs from 5637/shNC cells, while recombinant human VEGF 165 was added into CMs from 5637/shKLF5 cells. These CMs containing different levels of VEGFA were used to recruit HUVECs. B. HUVECs were serum-starved overnight, stimulated with SFM or CMs for 10 minutes before proteins harvest immediately. Phosphorylation of VEGFR2, Erk1/2 and Akt were evaluated by western blot. These data were representative of three independent experiments; * p

    Techniques Used: Recombinant, Western Blot

    39) Product Images from "Cancer metastasis and EGFR signaling is suppressed by amiodarone-induced versican V2"

    Article Title: Cancer metastasis and EGFR signaling is suppressed by amiodarone-induced versican V2

    Journal: Oncotarget

    doi:

    Amiodarone can induce Versican V2 expression, inhibit EGFR signaling and downstream AKT and ERK signaling Mouse tumor cells B16OVA, JC and 4T-1 cells A, C, E. and Human breast cancer cells MDA-MB-231 B, D, F. were treated with 15 uM or 20 μM Amiodarone (15A or 20A) for 24 hr and analyzed for Versican V1, V2 A, B, pEGFR (Tyr845), pEGFR (Tyr1173), EGFR C, D. pAKT (Ser473), AKT, pERK (T202/Y204) and ERK E, F. The relative intensities of each protein among control group (C) and Amiodarone-treated cells (15A) were as indicated. β-actin, and α-tubulin, were used as internal control.
    Figure Legend Snippet: Amiodarone can induce Versican V2 expression, inhibit EGFR signaling and downstream AKT and ERK signaling Mouse tumor cells B16OVA, JC and 4T-1 cells A, C, E. and Human breast cancer cells MDA-MB-231 B, D, F. were treated with 15 uM or 20 μM Amiodarone (15A or 20A) for 24 hr and analyzed for Versican V1, V2 A, B, pEGFR (Tyr845), pEGFR (Tyr1173), EGFR C, D. pAKT (Ser473), AKT, pERK (T202/Y204) and ERK E, F. The relative intensities of each protein among control group (C) and Amiodarone-treated cells (15A) were as indicated. β-actin, and α-tubulin, were used as internal control.

    Techniques Used: Expressing, Multiple Displacement Amplification

    40) Product Images from "NECAB3 Promotes Activation of Hypoxia-inducible factor-1 during Normoxia and Enhances Tumourigenicity of Cancer Cells"

    Article Title: NECAB3 Promotes Activation of Hypoxia-inducible factor-1 during Normoxia and Enhances Tumourigenicity of Cancer Cells

    Journal: Scientific Reports

    doi: 10.1038/srep22784

    NECAB3 depletion attenuates glycolysis in HT1080 cells. ( A ) Immunoblotting of NECAB3 and actin in whole cell lysates from control (shLacZ) and NECAB3-depleted (shNECAB3) HT1080 cells. This experiment was repeated three times with consistent results. ( B ) Immunoblotting of unphosphorylated and Ser473-phosphorylated AKT in whole cell lysates from control (shLacZ) and NECAB3-depleted cells. Consistent results were obtained from three experiments. ( C ) Pull-down assay of active, GTP-bound Ras in whole cell lysates from control (shLacZ) and NECAB3-depleted cells. Results were consistent in three experiments. ( D ) Immunoblotting of HIF-1α in nuclear lysates from control and NECB3-depleted cells. Lamin A/C was used as loading control. Results from three experiments were comparable. ( E ) Expression of SLC2A1 , PGK1 , PKM2 , PDK1 , LDHA , VEGFA , and ALDOA in control and NECAB3-depleted cells was analysed by real-time PCR and normalised to ACTB . ( F ) Glucose consumption and ( G ) lactate production in control and NECAB3-depleted HT1080 cells. ( H,I ) Lactate production in control and NECAB3-depleted A431 ( H ) and A549 cells ( I ). In ( E–I ) error bars indicate s.d. (n = 3), and data were analysed by t -test. * p
    Figure Legend Snippet: NECAB3 depletion attenuates glycolysis in HT1080 cells. ( A ) Immunoblotting of NECAB3 and actin in whole cell lysates from control (shLacZ) and NECAB3-depleted (shNECAB3) HT1080 cells. This experiment was repeated three times with consistent results. ( B ) Immunoblotting of unphosphorylated and Ser473-phosphorylated AKT in whole cell lysates from control (shLacZ) and NECAB3-depleted cells. Consistent results were obtained from three experiments. ( C ) Pull-down assay of active, GTP-bound Ras in whole cell lysates from control (shLacZ) and NECAB3-depleted cells. Results were consistent in three experiments. ( D ) Immunoblotting of HIF-1α in nuclear lysates from control and NECB3-depleted cells. Lamin A/C was used as loading control. Results from three experiments were comparable. ( E ) Expression of SLC2A1 , PGK1 , PKM2 , PDK1 , LDHA , VEGFA , and ALDOA in control and NECAB3-depleted cells was analysed by real-time PCR and normalised to ACTB . ( F ) Glucose consumption and ( G ) lactate production in control and NECAB3-depleted HT1080 cells. ( H,I ) Lactate production in control and NECAB3-depleted A431 ( H ) and A549 cells ( I ). In ( E–I ) error bars indicate s.d. (n = 3), and data were analysed by t -test. * p

    Techniques Used: Pull Down Assay, Expressing, Real-time Polymerase Chain Reaction

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    Article Title: Chronic exposure of interleukin‐13 suppress the induction of matrix metalloproteinase‐1 by tumour necrosis factor α in normal and scleroderma dermal fibroblasts through protein kinase B/Akt
    Article Snippet: .. Akt control cell extract (no. 9273; Cell Signaling) was used as a positive control. ..

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    Cell Signaling Technology Inc p akt
    PUMA induction by anti-EGFR antibodies is mediated by <t>p73</t> (A) DiFi cells transfected with control scrambled or p73 siRNA for 24 hr were re-plated and treated with 10 nM cetuximab (Cmab). Expression of p73 at 8 hr, and PUMA and cleaved (C) caspase-3 at 24 hr after cetuximab treatment was analyzed by western blotting. Cells without siRNA transfection and re-plating were used as the control for analyzing p73 at 8r after treatment. (B) Western blotting of indicated proteins in DiFi cells treated 10 nM cetuximab at the indicated time points. Phospho-p73 (p-p73, Y99); <t>phospho-AKT</t> (p-AKT, S473); phospho-ERK1/2 (p-ERK1/2, T202/Y204). (C) DiFi cells transfected with either a control empty vector or a HA-p73α construct were treated with 10 nM Cmab for the indicated times. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody with IgG as control, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (D) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected with siRNA as in (A) and treated with Cmab at the indicated doses for 72 hr. (E) Western blotting of indicated proteins in DiFi cells transfected with control empty vector or constitutively active AKT for 6 hr, and then treated with 10 nM cetuximab for 8 or 24 hr. (F) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected as in (E) and treated with Cmab at the indicated doses for 72 hr. (G) A model of PUMA induction by anti-EGFR antibodies.
    P Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 2853 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Immunohistochemistry of Qdot-GRP78 antibody in MDA-MB-231/GFP breast cells and LNCaP prostate cancer cells. ( A ) and ( B ) Fluorescence images of cells staining with Qdot625-GRP78 antibody. ( A ) control cells staining with unlabeled nanobeads; ( B ) MDA-MB-231/GFP cells staining with Qdot-GRP78 antibody; ( C ) Scanned confocal microscopy imaging in LNCaP prostate cancer cell, stained with anti-GRP78 mouse monoclonal antibody with secondary Goat anti-mouse IgG labeled with Alexa488(green)and Qdot-GRP78(red dots) and overlapped with two probes (yellow); ( D ) and ( E ) Internalization of Qdot-GRP78 antibody by MDA-MB-231/GFP cells. Cells were incubated with unlabelled nanobeads. ( D ) or Qdot-GRP78 antibody; ( E ) at 37 °C for 16 h. Cells were then washed with PBS and analyzed by fluorescence microscopy; ( F ) Scanned confocal microscopy imaging in MDA-MB-231/GFP cell (green), treated with control unlabelled beads; ( G ) Qdot-GRP78(red dots) was detected inside MDA-MB-231/GFP cell; ( H ) 3D reconstruction of confocal Z stack with 0.8-μM, GFP cell showing in green channel, while Qdot-GRP78 showing in red channel. Scale bar represents 20 μm; ( I ) Western blot analysis of GRP78 protein in Qdot-GRP78 antibody treated cells. Cells either treated with control (unlabelled Qdot) or Qdot-labeled antibody. Phosphorylated <t>Akt-ser473</t> protein was detected by an anti-anti-Akt-se473 antibody and Pan-Akt antibody was used as a loading control. The western blot represents three independent experiments. ( J ) MDA-MB-231/GFP cells were incubated with various concentrations of Qot-GRP78 antibody for 24h. Apoptotic nuclei or nuclear DNA strand breaks were visualized using Hoechst DNA dye H33342 (10). A minimum of 200 cells were counted in each sample and condensed or fragmented nuclei were expressed as a percentage of the total number of nuclei. Values are presented as mean ± S.D. of three determinations. * indicates significant difference ( p
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    PUMA induction by anti-EGFR antibodies is mediated by p73 (A) DiFi cells transfected with control scrambled or p73 siRNA for 24 hr were re-plated and treated with 10 nM cetuximab (Cmab). Expression of p73 at 8 hr, and PUMA and cleaved (C) caspase-3 at 24 hr after cetuximab treatment was analyzed by western blotting. Cells without siRNA transfection and re-plating were used as the control for analyzing p73 at 8r after treatment. (B) Western blotting of indicated proteins in DiFi cells treated 10 nM cetuximab at the indicated time points. Phospho-p73 (p-p73, Y99); phospho-AKT (p-AKT, S473); phospho-ERK1/2 (p-ERK1/2, T202/Y204). (C) DiFi cells transfected with either a control empty vector or a HA-p73α construct were treated with 10 nM Cmab for the indicated times. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody with IgG as control, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (D) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected with siRNA as in (A) and treated with Cmab at the indicated doses for 72 hr. (E) Western blotting of indicated proteins in DiFi cells transfected with control empty vector or constitutively active AKT for 6 hr, and then treated with 10 nM cetuximab for 8 or 24 hr. (F) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected as in (E) and treated with Cmab at the indicated doses for 72 hr. (G) A model of PUMA induction by anti-EGFR antibodies.

    Journal: Oncogene

    Article Title: Restoring PUMA induction overcomes KRAS-mediated resistance to anti-EGFR antibodies in colorectal cancer

    doi: 10.1038/s41388-018-0289-x

    Figure Lengend Snippet: PUMA induction by anti-EGFR antibodies is mediated by p73 (A) DiFi cells transfected with control scrambled or p73 siRNA for 24 hr were re-plated and treated with 10 nM cetuximab (Cmab). Expression of p73 at 8 hr, and PUMA and cleaved (C) caspase-3 at 24 hr after cetuximab treatment was analyzed by western blotting. Cells without siRNA transfection and re-plating were used as the control for analyzing p73 at 8r after treatment. (B) Western blotting of indicated proteins in DiFi cells treated 10 nM cetuximab at the indicated time points. Phospho-p73 (p-p73, Y99); phospho-AKT (p-AKT, S473); phospho-ERK1/2 (p-ERK1/2, T202/Y204). (C) DiFi cells transfected with either a control empty vector or a HA-p73α construct were treated with 10 nM Cmab for the indicated times. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody with IgG as control, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (D) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected with siRNA as in (A) and treated with Cmab at the indicated doses for 72 hr. (E) Western blotting of indicated proteins in DiFi cells transfected with control empty vector or constitutively active AKT for 6 hr, and then treated with 10 nM cetuximab for 8 or 24 hr. (F) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected as in (E) and treated with Cmab at the indicated doses for 72 hr. (G) A model of PUMA induction by anti-EGFR antibodies.

    Article Snippet: Western blotting Western blotting was performed as previously described [ ] using antibodies against: β-Actin (A5441, Sigma), cleaved caspase-3 (#9661, Cell Signaling, Danvers, MA, USA), cleaved caspase-9 (#9502, Cell Signaling), Mcl-1 (#559027, BD Biosciences, San Jose, CA, USA), Bax (#610983, BD Biosciences), Bid (#2002, Cell Signaling), Bcl-xL (#610212, BD Biosciences), Bim (#2819, Cell Signaling), Bcl-2 (#M0887, Agilent DAKO, Santa Clara, CA, USA), cytochrome c (sc-7159, Santa Cruz Biotechnology, Santa Cruz, CA, USA), COX IV (A21348, Invitrogen), PUMA [ ], Bak (#06–536, EMD Millipore), Noxa (#OP180, EMD Millipore), p73 (A300–126A, Bethyl Laboratories, Montgomery, TX, USA), p-p73 (#4665, Cell Signaling), p-AKT (#4058, Cell Signaling), total AKT (#9272, Cell Signaling), p-ERK1/2 (#4376, Cell Signaling), total ERK1/2 (#9102, Cell Signaling), p-FoxO3A (#9464, Cell Signaling), total FoxO3A (07–702, EMD Millipore), p53 (sc-126, Santa Cruz), p-EGFR (#2234, Cell Signaling), total EGFR (#610016, BD Biosciences), KRAS (sc-30, Santa Cruz), p-Aurora A/B/C (#2914, Cell Signaling), total Aurora A (#4718, Cell Signaling), total Aurora B (#3094, Cell Signaling), and HA (#12CA5, Roche, Indianapolis, IN, USA).

    Techniques: Transfection, Expressing, Western Blot, Plasmid Preparation, Construct, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining

    KRAS -mediated resistance to anti-EGFR antibodies abrogates PUMA induction (A) MTS analysis of parental (red) and cetuximab-resistant (Cmab-R, black) DiFi cells treated with cetuximab (Cmab) or panitumumab (Pmab) at the indicated doses for 72 hr. (B) Western blotting of cleaved (C) caspase-3 in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab or Pmab for 24 hr. (C) Western blotting of indicated proteins in the parental and Cmab-R DiFi cells. Lysates of Cmab-R DiFi cells were prepared from cells cultured in medium with 10 nM cetuximab (Cmab+), or without cetuximab (Cmab-) for 6 days. p-EGFR (Y1068); p-AKT (S473); p-ERK1/2 (T202/Y204). (D) Western blotting of indicated Bcl-2 family proteins in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab for 24 hr. (E) Western blotting of phosphorylated (p-p73, Y99) and total p73 in the parental and Cmab-R DiFi cells treated with 10 nM Cmab for 8 hr. (F) Parental and Cmab-R DiFi cells transfected with control empty or HA-p73α-expressing vector were treated with 10 nM cetuximab for 8 hr. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (G) Parental and Cmab-R DiFi cells were infected with EGFP-PUMA-expressing adenovirus (Ad-PUMA) at the indicated MOI for 24 hr. Upper, analysis of apoptosis by nuclear fragmentation; lower , western blotting of PUMA. Results were expressed as means ± s.e.m. of triplicates in two independent experiments. *** P

    Journal: Oncogene

    Article Title: Restoring PUMA induction overcomes KRAS-mediated resistance to anti-EGFR antibodies in colorectal cancer

    doi: 10.1038/s41388-018-0289-x

    Figure Lengend Snippet: KRAS -mediated resistance to anti-EGFR antibodies abrogates PUMA induction (A) MTS analysis of parental (red) and cetuximab-resistant (Cmab-R, black) DiFi cells treated with cetuximab (Cmab) or panitumumab (Pmab) at the indicated doses for 72 hr. (B) Western blotting of cleaved (C) caspase-3 in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab or Pmab for 24 hr. (C) Western blotting of indicated proteins in the parental and Cmab-R DiFi cells. Lysates of Cmab-R DiFi cells were prepared from cells cultured in medium with 10 nM cetuximab (Cmab+), or without cetuximab (Cmab-) for 6 days. p-EGFR (Y1068); p-AKT (S473); p-ERK1/2 (T202/Y204). (D) Western blotting of indicated Bcl-2 family proteins in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab for 24 hr. (E) Western blotting of phosphorylated (p-p73, Y99) and total p73 in the parental and Cmab-R DiFi cells treated with 10 nM Cmab for 8 hr. (F) Parental and Cmab-R DiFi cells transfected with control empty or HA-p73α-expressing vector were treated with 10 nM cetuximab for 8 hr. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (G) Parental and Cmab-R DiFi cells were infected with EGFP-PUMA-expressing adenovirus (Ad-PUMA) at the indicated MOI for 24 hr. Upper, analysis of apoptosis by nuclear fragmentation; lower , western blotting of PUMA. Results were expressed as means ± s.e.m. of triplicates in two independent experiments. *** P

    Article Snippet: Western blotting Western blotting was performed as previously described [ ] using antibodies against: β-Actin (A5441, Sigma), cleaved caspase-3 (#9661, Cell Signaling, Danvers, MA, USA), cleaved caspase-9 (#9502, Cell Signaling), Mcl-1 (#559027, BD Biosciences, San Jose, CA, USA), Bax (#610983, BD Biosciences), Bid (#2002, Cell Signaling), Bcl-xL (#610212, BD Biosciences), Bim (#2819, Cell Signaling), Bcl-2 (#M0887, Agilent DAKO, Santa Clara, CA, USA), cytochrome c (sc-7159, Santa Cruz Biotechnology, Santa Cruz, CA, USA), COX IV (A21348, Invitrogen), PUMA [ ], Bak (#06–536, EMD Millipore), Noxa (#OP180, EMD Millipore), p73 (A300–126A, Bethyl Laboratories, Montgomery, TX, USA), p-p73 (#4665, Cell Signaling), p-AKT (#4058, Cell Signaling), total AKT (#9272, Cell Signaling), p-ERK1/2 (#4376, Cell Signaling), total ERK1/2 (#9102, Cell Signaling), p-FoxO3A (#9464, Cell Signaling), total FoxO3A (07–702, EMD Millipore), p53 (sc-126, Santa Cruz), p-EGFR (#2234, Cell Signaling), total EGFR (#610016, BD Biosciences), KRAS (sc-30, Santa Cruz), p-Aurora A/B/C (#2914, Cell Signaling), total Aurora A (#4718, Cell Signaling), total Aurora B (#3094, Cell Signaling), and HA (#12CA5, Roche, Indianapolis, IN, USA).

    Techniques: Western Blot, Cell Culture, Transfection, Expressing, Plasmid Preparation, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Infection

    The downregulation of CDC20 inhibited the phenylephrine (PE)-induced hypertrophic response in cultured cardiomyocytes. (A) Immunoblotting analysis of CDC20 in neonatal rat cardiomyocytes infected with adenovirus siCDC20 or siControl (n=3). (B) Representative images of double immunostaining (red for α-actinin, blue for DAPI) of NRCMs after 24 hours of PE (100 nM) (left). Scale bar: 50 μm. Quantification of the myocyte surface area (n=3, 150 cells counted per experiment; right). (C) qPCR analysis of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) mRNA levels in NRCMs treated as described in (B) (n=3). (D) Immunoblot analysis of AKT, ERK1/2, p38 and GAPDH in NRCMs treated as described in (B) (n=3); * P

    Journal: Theranostics

    Article Title: CDC20 regulates cardiac hypertrophy via targeting LC3-dependent autophagy

    doi: 10.7150/thno.27706

    Figure Lengend Snippet: The downregulation of CDC20 inhibited the phenylephrine (PE)-induced hypertrophic response in cultured cardiomyocytes. (A) Immunoblotting analysis of CDC20 in neonatal rat cardiomyocytes infected with adenovirus siCDC20 or siControl (n=3). (B) Representative images of double immunostaining (red for α-actinin, blue for DAPI) of NRCMs after 24 hours of PE (100 nM) (left). Scale bar: 50 μm. Quantification of the myocyte surface area (n=3, 150 cells counted per experiment; right). (C) qPCR analysis of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) mRNA levels in NRCMs treated as described in (B) (n=3). (D) Immunoblot analysis of AKT, ERK1/2, p38 and GAPDH in NRCMs treated as described in (B) (n=3); * P

    Article Snippet: Antibodies and reagents Antibodies: LC3B (Sigma; L7532), ATG5 (Cell Signalling; 12994), AKT (Cell Signalling; 9272), p-AKT (Cell Signalling; 4060S), ERK1/2(Cell Signalling; 9102), p-ERK1/2 (Cell Signalling; 9101), Beclin-1 (Cell Signalling; 3738), horseradish peroxidase-linked anti-mouse or anti-rabbit IgG (Cell Signalling; 7074S).

    Techniques: Cell Culture, Infection, Double Immunostaining, Real-time Polymerase Chain Reaction, Aqueous Normal-phase Chromatography

    The knockdown of CDC20 suppresses hypertrophy in vivo . (A) Wild-type (WT) mice injected with rAAV9-siZsGreen or rAAV9-siCDC20 were subjected to sham operation or TAC for 5 weeks. Echocardiographic assessment of ejection fraction (EF%) and fractional shortening (FS%) (n=6). (B) H E staining of heart sections (upper). Scale bar 0.5 cm. The ratios of heart weight to tibia length (HW/TL) and body weight (HW/BW) (lower) (n=6). (C) Heart cross-sections were stained with TRITC-labeled wheat germ agglutinin (WGA, upper panel). Scale bar: 10 μm. Quantification of the relative myocyte cross-sectional area (n=6, 200 cells counted per heart; lower panel). (D) qPCR analysis of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) mRNA levels in the hearts of rAAV9-siZsGreen- and rAAV9-siCDC20-treated mice following sham operation or TAC (n=6). (E) Representative images of Masson's Trichrome (upper) and DHE staining (middle) of ventricular sections. Scale bar: 50 μm. Quantification of the relative fibrotic area and ROS fluorescence intensity (n=6, lower). (F) Immunoblotting analysis of p-AKT, AKT, p-ERK1/2, ERK1/2, NOX4 and GAPDH in heart samples (n=6). * P

    Journal: Theranostics

    Article Title: CDC20 regulates cardiac hypertrophy via targeting LC3-dependent autophagy

    doi: 10.7150/thno.27706

    Figure Lengend Snippet: The knockdown of CDC20 suppresses hypertrophy in vivo . (A) Wild-type (WT) mice injected with rAAV9-siZsGreen or rAAV9-siCDC20 were subjected to sham operation or TAC for 5 weeks. Echocardiographic assessment of ejection fraction (EF%) and fractional shortening (FS%) (n=6). (B) H E staining of heart sections (upper). Scale bar 0.5 cm. The ratios of heart weight to tibia length (HW/TL) and body weight (HW/BW) (lower) (n=6). (C) Heart cross-sections were stained with TRITC-labeled wheat germ agglutinin (WGA, upper panel). Scale bar: 10 μm. Quantification of the relative myocyte cross-sectional area (n=6, 200 cells counted per heart; lower panel). (D) qPCR analysis of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) mRNA levels in the hearts of rAAV9-siZsGreen- and rAAV9-siCDC20-treated mice following sham operation or TAC (n=6). (E) Representative images of Masson's Trichrome (upper) and DHE staining (middle) of ventricular sections. Scale bar: 50 μm. Quantification of the relative fibrotic area and ROS fluorescence intensity (n=6, lower). (F) Immunoblotting analysis of p-AKT, AKT, p-ERK1/2, ERK1/2, NOX4 and GAPDH in heart samples (n=6). * P

    Article Snippet: Antibodies and reagents Antibodies: LC3B (Sigma; L7532), ATG5 (Cell Signalling; 12994), AKT (Cell Signalling; 9272), p-AKT (Cell Signalling; 4060S), ERK1/2(Cell Signalling; 9102), p-ERK1/2 (Cell Signalling; 9101), Beclin-1 (Cell Signalling; 3738), horseradish peroxidase-linked anti-mouse or anti-rabbit IgG (Cell Signalling; 7074S).

    Techniques: In Vivo, Mouse Assay, Injection, Staining, Labeling, Whole Genome Amplification, Real-time Polymerase Chain Reaction, Aqueous Normal-phase Chromatography, Fluorescence

    Knockdown of LC3 reversed the reduction of cardiomyocyte size after deletion of CDC20 in vitro . (A) Representative images of double immunostaining (green for α-actinin) of NRCMs transfected with siRNA-CDC20 or siRNA-control with or without Ad-siLC3 treatment after 24 hours of Ang II stimulation (left). Scale bar 50 μm. Quantification of the myocyte surface area (right) (n=3). (B) Representative Western blot analyses of p-AKT, AKT, p-ERK1/2, ERK1/2, NOX4, LC3 I/II and GAPDH in NRCMs infected with siRNA-control or siRNA-CDC20 with or without Ad-siLC3 treatment after 24 hours of Ang II stimulation (n=3). C, A functional link between CDC20 and LC3-dependent autophagy in hypertrophy. ** P

    Journal: Theranostics

    Article Title: CDC20 regulates cardiac hypertrophy via targeting LC3-dependent autophagy

    doi: 10.7150/thno.27706

    Figure Lengend Snippet: Knockdown of LC3 reversed the reduction of cardiomyocyte size after deletion of CDC20 in vitro . (A) Representative images of double immunostaining (green for α-actinin) of NRCMs transfected with siRNA-CDC20 or siRNA-control with or without Ad-siLC3 treatment after 24 hours of Ang II stimulation (left). Scale bar 50 μm. Quantification of the myocyte surface area (right) (n=3). (B) Representative Western blot analyses of p-AKT, AKT, p-ERK1/2, ERK1/2, NOX4, LC3 I/II and GAPDH in NRCMs infected with siRNA-control or siRNA-CDC20 with or without Ad-siLC3 treatment after 24 hours of Ang II stimulation (n=3). C, A functional link between CDC20 and LC3-dependent autophagy in hypertrophy. ** P

    Article Snippet: Antibodies and reagents Antibodies: LC3B (Sigma; L7532), ATG5 (Cell Signalling; 12994), AKT (Cell Signalling; 9272), p-AKT (Cell Signalling; 4060S), ERK1/2(Cell Signalling; 9102), p-ERK1/2 (Cell Signalling; 9101), Beclin-1 (Cell Signalling; 3738), horseradish peroxidase-linked anti-mouse or anti-rabbit IgG (Cell Signalling; 7074S).

    Techniques: In Vitro, Double Immunostaining, Transfection, Western Blot, Infection, Functional Assay

    The effect of MDHB on AKT, GSK3β and β-catenin. MDHB inhibited phosphorylation of AKT in the differentiation of neural stem cells, activated phosphorylation of GSK3β at tyrosine 216 (Y216), and downregulated transcription factor β-catenin. (A,B) Western blot analyses of proteins extracted from MDHB-treated neural stem cell in differentiation, (C–H) quantification of protein blots is shown, GAPDH serves as protein loading control. Each point represents the mean relative protein level of each group ( ∗ P

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Methyl 3,4-Dihydroxybenzoate Induces Neural Stem Cells to Differentiate Into Cholinergic Neurons in vitro

    doi: 10.3389/fncel.2018.00478

    Figure Lengend Snippet: The effect of MDHB on AKT, GSK3β and β-catenin. MDHB inhibited phosphorylation of AKT in the differentiation of neural stem cells, activated phosphorylation of GSK3β at tyrosine 216 (Y216), and downregulated transcription factor β-catenin. (A,B) Western blot analyses of proteins extracted from MDHB-treated neural stem cell in differentiation, (C–H) quantification of protein blots is shown, GAPDH serves as protein loading control. Each point represents the mean relative protein level of each group ( ∗ P

    Article Snippet: The expressions of β-III-tubulin, GFAP, AKT (CST), p-AKT (CST), GSK3β (CST), p-ser9-GSK3β (CST), p-try-GSK3β (Thermofisher) and β-catenin (CST) were determined via calculating their density ratio to the GAPDH band.

    Techniques: Western Blot

    Immunohistochemistry of Qdot-GRP78 antibody in MDA-MB-231/GFP breast cells and LNCaP prostate cancer cells. ( A ) and ( B ) Fluorescence images of cells staining with Qdot625-GRP78 antibody. ( A ) control cells staining with unlabeled nanobeads; ( B ) MDA-MB-231/GFP cells staining with Qdot-GRP78 antibody; ( C ) Scanned confocal microscopy imaging in LNCaP prostate cancer cell, stained with anti-GRP78 mouse monoclonal antibody with secondary Goat anti-mouse IgG labeled with Alexa488(green)and Qdot-GRP78(red dots) and overlapped with two probes (yellow); ( D ) and ( E ) Internalization of Qdot-GRP78 antibody by MDA-MB-231/GFP cells. Cells were incubated with unlabelled nanobeads. ( D ) or Qdot-GRP78 antibody; ( E ) at 37 °C for 16 h. Cells were then washed with PBS and analyzed by fluorescence microscopy; ( F ) Scanned confocal microscopy imaging in MDA-MB-231/GFP cell (green), treated with control unlabelled beads; ( G ) Qdot-GRP78(red dots) was detected inside MDA-MB-231/GFP cell; ( H ) 3D reconstruction of confocal Z stack with 0.8-μM, GFP cell showing in green channel, while Qdot-GRP78 showing in red channel. Scale bar represents 20 μm; ( I ) Western blot analysis of GRP78 protein in Qdot-GRP78 antibody treated cells. Cells either treated with control (unlabelled Qdot) or Qdot-labeled antibody. Phosphorylated Akt-ser473 protein was detected by an anti-anti-Akt-se473 antibody and Pan-Akt antibody was used as a loading control. The western blot represents three independent experiments. ( J ) MDA-MB-231/GFP cells were incubated with various concentrations of Qot-GRP78 antibody for 24h. Apoptotic nuclei or nuclear DNA strand breaks were visualized using Hoechst DNA dye H33342 (10). A minimum of 200 cells were counted in each sample and condensed or fragmented nuclei were expressed as a percentage of the total number of nuclei. Values are presented as mean ± S.D. of three determinations. * indicates significant difference ( p

    Journal: Molecules

    Article Title: Quantum Dot- Conjugated Anti-GRP78 scFv Inhibits Cancer Growth in Mice

    doi: 10.3390/molecules17010796

    Figure Lengend Snippet: Immunohistochemistry of Qdot-GRP78 antibody in MDA-MB-231/GFP breast cells and LNCaP prostate cancer cells. ( A ) and ( B ) Fluorescence images of cells staining with Qdot625-GRP78 antibody. ( A ) control cells staining with unlabeled nanobeads; ( B ) MDA-MB-231/GFP cells staining with Qdot-GRP78 antibody; ( C ) Scanned confocal microscopy imaging in LNCaP prostate cancer cell, stained with anti-GRP78 mouse monoclonal antibody with secondary Goat anti-mouse IgG labeled with Alexa488(green)and Qdot-GRP78(red dots) and overlapped with two probes (yellow); ( D ) and ( E ) Internalization of Qdot-GRP78 antibody by MDA-MB-231/GFP cells. Cells were incubated with unlabelled nanobeads. ( D ) or Qdot-GRP78 antibody; ( E ) at 37 °C for 16 h. Cells were then washed with PBS and analyzed by fluorescence microscopy; ( F ) Scanned confocal microscopy imaging in MDA-MB-231/GFP cell (green), treated with control unlabelled beads; ( G ) Qdot-GRP78(red dots) was detected inside MDA-MB-231/GFP cell; ( H ) 3D reconstruction of confocal Z stack with 0.8-μM, GFP cell showing in green channel, while Qdot-GRP78 showing in red channel. Scale bar represents 20 μm; ( I ) Western blot analysis of GRP78 protein in Qdot-GRP78 antibody treated cells. Cells either treated with control (unlabelled Qdot) or Qdot-labeled antibody. Phosphorylated Akt-ser473 protein was detected by an anti-anti-Akt-se473 antibody and Pan-Akt antibody was used as a loading control. The western blot represents three independent experiments. ( J ) MDA-MB-231/GFP cells were incubated with various concentrations of Qot-GRP78 antibody for 24h. Apoptotic nuclei or nuclear DNA strand breaks were visualized using Hoechst DNA dye H33342 (10). A minimum of 200 cells were counted in each sample and condensed or fragmented nuclei were expressed as a percentage of the total number of nuclei. Values are presented as mean ± S.D. of three determinations. * indicates significant difference ( p

    Article Snippet: Anti-phosphot-Akt (ser473) and anti-Akt (pan) were from the Cell Signaling Technology, Inc (Danvers, USA).

    Techniques: Immunohistochemistry, Multiple Displacement Amplification, Fluorescence, Staining, Confocal Microscopy, Imaging, Labeling, Incubation, Microscopy, Western Blot