Structured Review

Santa Cruz Biotechnology anti akt
RasGRF1 is involved in chemokine and growth factor receptor signaling. (A), Effect of RasGRF1 down-regulation on activation of intracellular signaling in ARMS cells. Phosphorylation of <t>p42/44</t> MAPK and <t>AKT</t> in RH30-derived cell lines was stimulated for 5 min by SDF-1 (300 ng/ml), I-TAC (100 ng/ml), HGF (100 ng/ml), IGF-II (100 ng/ml), and insulin (10 ng/ml). The experiment was repeated three times with similar results. A representative result is shown. (B), RasGRF1 phosphorylation after stimulation with chemokines and growth factors. RasGRF1 protein phophorylated at Ser929 was detected by western blot analysis after stimulation for 5 min by SDF-1 (300 ng/ml), I-TAC (100 ng/ml), HGF (100 ng/ml), IGF-II (100 ng/ml), and insulin (10 ng/ml). (C), Effect of RasGRF1 down-regulation on Ras activation. A Ras pull-down assay was performed on two RH30-derived cell lines (RH30scr and RH30 RasGRF1-kd). The cells were stimulated for 5 min with SDF-1 (300 ng/ml) or IGF-II (100 ng/ml). Ras-GTP was precipitated by Raf-1 RBD agarose conjugate and detected by Ras antibody clone RAS10 (Millipore). The same antibody was used to detect total Ras protein (p21 H-, K- and N-Ras). Western blots were analyzed by densitometry (right side). The experiment was repeated three times with similar results. A representative result is shown. (D), Effect of RasGRF1 down-regulation on paxillin expression and actin cytoskeleton. Staining of paxillin and actin was performed on RH30scr, RH30 RasGRF1-kd, RH18scr and RH18 RasGRF-kd cell lines. The experiment was repeated three times and representative results are shown.
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1) Product Images from "RasGRF1 regulates proliferation and metastatic behavior of human alveolar rhabdomyosarcomas"

Article Title: RasGRF1 regulates proliferation and metastatic behavior of human alveolar rhabdomyosarcomas

Journal: International Journal of Oncology

doi: 10.3892/ijo.2012.1536

RasGRF1 is involved in chemokine and growth factor receptor signaling. (A), Effect of RasGRF1 down-regulation on activation of intracellular signaling in ARMS cells. Phosphorylation of p42/44 MAPK and AKT in RH30-derived cell lines was stimulated for 5 min by SDF-1 (300 ng/ml), I-TAC (100 ng/ml), HGF (100 ng/ml), IGF-II (100 ng/ml), and insulin (10 ng/ml). The experiment was repeated three times with similar results. A representative result is shown. (B), RasGRF1 phosphorylation after stimulation with chemokines and growth factors. RasGRF1 protein phophorylated at Ser929 was detected by western blot analysis after stimulation for 5 min by SDF-1 (300 ng/ml), I-TAC (100 ng/ml), HGF (100 ng/ml), IGF-II (100 ng/ml), and insulin (10 ng/ml). (C), Effect of RasGRF1 down-regulation on Ras activation. A Ras pull-down assay was performed on two RH30-derived cell lines (RH30scr and RH30 RasGRF1-kd). The cells were stimulated for 5 min with SDF-1 (300 ng/ml) or IGF-II (100 ng/ml). Ras-GTP was precipitated by Raf-1 RBD agarose conjugate and detected by Ras antibody clone RAS10 (Millipore). The same antibody was used to detect total Ras protein (p21 H-, K- and N-Ras). Western blots were analyzed by densitometry (right side). The experiment was repeated three times with similar results. A representative result is shown. (D), Effect of RasGRF1 down-regulation on paxillin expression and actin cytoskeleton. Staining of paxillin and actin was performed on RH30scr, RH30 RasGRF1-kd, RH18scr and RH18 RasGRF-kd cell lines. The experiment was repeated three times and representative results are shown.
Figure Legend Snippet: RasGRF1 is involved in chemokine and growth factor receptor signaling. (A), Effect of RasGRF1 down-regulation on activation of intracellular signaling in ARMS cells. Phosphorylation of p42/44 MAPK and AKT in RH30-derived cell lines was stimulated for 5 min by SDF-1 (300 ng/ml), I-TAC (100 ng/ml), HGF (100 ng/ml), IGF-II (100 ng/ml), and insulin (10 ng/ml). The experiment was repeated three times with similar results. A representative result is shown. (B), RasGRF1 phosphorylation after stimulation with chemokines and growth factors. RasGRF1 protein phophorylated at Ser929 was detected by western blot analysis after stimulation for 5 min by SDF-1 (300 ng/ml), I-TAC (100 ng/ml), HGF (100 ng/ml), IGF-II (100 ng/ml), and insulin (10 ng/ml). (C), Effect of RasGRF1 down-regulation on Ras activation. A Ras pull-down assay was performed on two RH30-derived cell lines (RH30scr and RH30 RasGRF1-kd). The cells were stimulated for 5 min with SDF-1 (300 ng/ml) or IGF-II (100 ng/ml). Ras-GTP was precipitated by Raf-1 RBD agarose conjugate and detected by Ras antibody clone RAS10 (Millipore). The same antibody was used to detect total Ras protein (p21 H-, K- and N-Ras). Western blots were analyzed by densitometry (right side). The experiment was repeated three times with similar results. A representative result is shown. (D), Effect of RasGRF1 down-regulation on paxillin expression and actin cytoskeleton. Staining of paxillin and actin was performed on RH30scr, RH30 RasGRF1-kd, RH18scr and RH18 RasGRF-kd cell lines. The experiment was repeated three times and representative results are shown.

Techniques Used: Activation Assay, Derivative Assay, Western Blot, Pull Down Assay, Expressing, Staining

2) Product Images from "Function of the Nucleotide Exchange Activity of Vav1 in T cell Development and Activation"

Article Title: Function of the Nucleotide Exchange Activity of Vav1 in T cell Development and Activation

Journal: Science signaling

doi: 10.1126/scisignal.2000420

The GEF activity of Vav1 is required for a subset of TCR-induced signaling pathways. ( A , B ) Western blots of total cytoplasmic extracts of naïve CD4 + CD44 lo lymph node T cells from mice of the indicated genotypes that were stimulated through CD3ε and CD28 for the indicated times. Western blots were incubated with antibodies against phosphorylated forms of ERK2, PKD1, Akt, and LAT, and with antibodies against total ERK2, PKD1, Akt, and LAT. The graphs on the right show the relative abundance of phosphorylated forms of ERK2, PKD1, Akt, and LAT, as determined from a ratio of the intensity of the bands of phosphorylated proteins to those of total proteins. Comparison of Vav1 AA/AA and Vav1 −/− T cells with (A) WT or (B) Vav1 +/− T cells. ( C ) Western blots of active Rac1-GTP pulled down with a GST-Pak1-RBD fusion protein and of total cytoplasmic lysates from CD4 + T cells stimulated through CD3ε and CD28 for the indicated times. Western blots were incubated with an antibody against Rac1. Graph shows mean ± SEM ratios of Rac1-GTP to total Rac1 of three independent experiments, normalized as described in Materials and Methods. ( D ) Western blots and graphs (presented as described for panel A) of WT naïve CD4 + lymph node T cells treated with either 20 μM or 40 μM of the Rac inhibitor EHT 1864 or the inactive control compound EHT 4063.
Figure Legend Snippet: The GEF activity of Vav1 is required for a subset of TCR-induced signaling pathways. ( A , B ) Western blots of total cytoplasmic extracts of naïve CD4 + CD44 lo lymph node T cells from mice of the indicated genotypes that were stimulated through CD3ε and CD28 for the indicated times. Western blots were incubated with antibodies against phosphorylated forms of ERK2, PKD1, Akt, and LAT, and with antibodies against total ERK2, PKD1, Akt, and LAT. The graphs on the right show the relative abundance of phosphorylated forms of ERK2, PKD1, Akt, and LAT, as determined from a ratio of the intensity of the bands of phosphorylated proteins to those of total proteins. Comparison of Vav1 AA/AA and Vav1 −/− T cells with (A) WT or (B) Vav1 +/− T cells. ( C ) Western blots of active Rac1-GTP pulled down with a GST-Pak1-RBD fusion protein and of total cytoplasmic lysates from CD4 + T cells stimulated through CD3ε and CD28 for the indicated times. Western blots were incubated with an antibody against Rac1. Graph shows mean ± SEM ratios of Rac1-GTP to total Rac1 of three independent experiments, normalized as described in Materials and Methods. ( D ) Western blots and graphs (presented as described for panel A) of WT naïve CD4 + lymph node T cells treated with either 20 μM or 40 μM of the Rac inhibitor EHT 1864 or the inactive control compound EHT 4063.

Techniques Used: Activity Assay, Western Blot, Mouse Assay, Incubation

3) Product Images from "S100A8 is a Novel Therapeutic Target for Anaplastic Thyroid Carcinoma"

Article Title: S100A8 is a Novel Therapeutic Target for Anaplastic Thyroid Carcinoma

Journal: The Journal of Clinical Endocrinology and Metabolism

doi: 10.1210/jc.2014-2988

Screening of signaling pathways activated by S100A8. A, Immunofluorescent staining of S100A8 and RAGE in THJ-11T cells. B, FACS analysis of S100A8 and RAGE in THJ-11T cells. Red lines represent isotype controls, blue lines show positive staining for S100A8 or RAGE. C, Immunoprecipitation experiments demonstrated that S100A8 was physically associated with RAGE. THJ-11T cell lysates were immunoprecipitated with anti-S100A8 or an irrelevant control rabbit IgG. Whole-cell lysates and immunoprecipitated proteins were separated on SDS-PAGE and immunoblotted with anti-RAGE antibodies. Results depicted are representative of three experiments. D, Cells were either left untreated or treated with 20 μg/mL of S100A8 for 3 h. Whole-cell lysates were probed with a human phosphokinase array. Proteins showing increased phosphorylation upon S100A8 treatment are highlighted. E, An analysis of the mean pixel density of highlighted spots (p38, ERK1/2, JNK) of the array. F, Cells were starved overnight followed by treatment with increasing S100A8 protein concentrations (0.1 to 20 μg/mL) for 1 h. Whole-cell lysates were examined by Western blot analysis using phosphospecific antibodies, pERK (T202/Y204), ERK, pJNK (T183/Y185), JNK, pp38 (T180/Y182), p38, pAKT (S473), and AKT. Data are representative of two independent experiments.
Figure Legend Snippet: Screening of signaling pathways activated by S100A8. A, Immunofluorescent staining of S100A8 and RAGE in THJ-11T cells. B, FACS analysis of S100A8 and RAGE in THJ-11T cells. Red lines represent isotype controls, blue lines show positive staining for S100A8 or RAGE. C, Immunoprecipitation experiments demonstrated that S100A8 was physically associated with RAGE. THJ-11T cell lysates were immunoprecipitated with anti-S100A8 or an irrelevant control rabbit IgG. Whole-cell lysates and immunoprecipitated proteins were separated on SDS-PAGE and immunoblotted with anti-RAGE antibodies. Results depicted are representative of three experiments. D, Cells were either left untreated or treated with 20 μg/mL of S100A8 for 3 h. Whole-cell lysates were probed with a human phosphokinase array. Proteins showing increased phosphorylation upon S100A8 treatment are highlighted. E, An analysis of the mean pixel density of highlighted spots (p38, ERK1/2, JNK) of the array. F, Cells were starved overnight followed by treatment with increasing S100A8 protein concentrations (0.1 to 20 μg/mL) for 1 h. Whole-cell lysates were examined by Western blot analysis using phosphospecific antibodies, pERK (T202/Y204), ERK, pJNK (T183/Y185), JNK, pp38 (T180/Y182), p38, pAKT (S473), and AKT. Data are representative of two independent experiments.

Techniques Used: Staining, FACS, Immunoprecipitation, SDS Page, Western Blot

4) Product Images from "Centrosomal Protein of 55 Regulates Glucose Metabolism, Proliferation and Apoptosis of Glioma Cells via the Akt/mTOR Signaling Pathway"

Article Title: Centrosomal Protein of 55 Regulates Glucose Metabolism, Proliferation and Apoptosis of Glioma Cells via the Akt/mTOR Signaling Pathway

Journal: Journal of Cancer

doi: 10.7150/jca.15497

CEP55 regulates the Akt/mTOR signaling pathway. (A) Protein levels of p-Akt, total Akt, p-mTOR and total mTOR as determined by Western blot analysis in U87 and T98G cells transfected with control siRNA or siRNA targeting CEP55. GAPDH was used as the loading control. (B) Protein levels of Akt downstream targets (BAD, Caspase-9, p27 and GSK-3β) as determined by Western blot analysis in U87 and T98G cells transfected with control siRNA or siRNA targeting CEP55.
Figure Legend Snippet: CEP55 regulates the Akt/mTOR signaling pathway. (A) Protein levels of p-Akt, total Akt, p-mTOR and total mTOR as determined by Western blot analysis in U87 and T98G cells transfected with control siRNA or siRNA targeting CEP55. GAPDH was used as the loading control. (B) Protein levels of Akt downstream targets (BAD, Caspase-9, p27 and GSK-3β) as determined by Western blot analysis in U87 and T98G cells transfected with control siRNA or siRNA targeting CEP55.

Techniques Used: Western Blot, Transfection

Knockdown of CEP55 inhibits glioma development via suppressing Akt/mTOR signaling. (A) Relative glucose uptake, (B) cell proliferation and (C) cell viability in U87 and T98G cells transfected with control siRNA or siRNA targeting CEP55 treated with or without Akt inhibitor MK-2206 or mTOR inhibitor rapamycin.
Figure Legend Snippet: Knockdown of CEP55 inhibits glioma development via suppressing Akt/mTOR signaling. (A) Relative glucose uptake, (B) cell proliferation and (C) cell viability in U87 and T98G cells transfected with control siRNA or siRNA targeting CEP55 treated with or without Akt inhibitor MK-2206 or mTOR inhibitor rapamycin.

Techniques Used: Transfection

5) Product Images from "Activation of the MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis protects MG-63 osteosarcoma cells against 15d-PGJ2-mediated cell death"

Article Title: Activation of the MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis protects MG-63 osteosarcoma cells against 15d-PGJ2-mediated cell death

Journal: Biochemical pharmacology

doi: 10.1016/j.bcp.2016.01.011

The chemical structure of (dh-)15d-PGJ 2 and possible activation cascades. (A) The chemical structure of 15d-PGJ 2 and dh-15d-PGJ 2 (9,10-dihydro-15d-PGJ 2 , * indicates electrophilic carbon atom). (B–F) MG-63 cells were stimulated with dh-15d-PGJ 2 (20 μM) or 15d-PGJ 2 (20 μM, used as a positive control) for (B) 15 min to follow phosphorylation of p38 MAPK, (C) 1 h to follow phosphorylation of Akt, (D) 6 h to follow Nrf2 and Egr1 expression, (E) 6 h to follow HO-1 and GCLc expression or (F) 24 h to follow cleavage of pro-caspase and PARP. For Western blot analysis total protein lysates were subjected to SDS–PAGE. (B) Total p38 MAPK, (C) total Akt, and (D–F) β-actin were used as loading controls. One representative blot out of two is shown. Pro-caspase-3 (p-Caspase-3, molecular mass: 35 kDa); cleaved caspase-3 (c-Caspase-3; molecular mass: 19 and 17 kDa). Poly (ADP-ribose) polymerase (PARP; molecular mass: 116 kDa; cleaved PARP; molecular mass: 89 kDa). As membranes for Nrf2/Egr1 (D) were stripped and reprobed with anti-HO-1/-GCLc antibodies (E), the same β-actin blot is shown.
Figure Legend Snippet: The chemical structure of (dh-)15d-PGJ 2 and possible activation cascades. (A) The chemical structure of 15d-PGJ 2 and dh-15d-PGJ 2 (9,10-dihydro-15d-PGJ 2 , * indicates electrophilic carbon atom). (B–F) MG-63 cells were stimulated with dh-15d-PGJ 2 (20 μM) or 15d-PGJ 2 (20 μM, used as a positive control) for (B) 15 min to follow phosphorylation of p38 MAPK, (C) 1 h to follow phosphorylation of Akt, (D) 6 h to follow Nrf2 and Egr1 expression, (E) 6 h to follow HO-1 and GCLc expression or (F) 24 h to follow cleavage of pro-caspase and PARP. For Western blot analysis total protein lysates were subjected to SDS–PAGE. (B) Total p38 MAPK, (C) total Akt, and (D–F) β-actin were used as loading controls. One representative blot out of two is shown. Pro-caspase-3 (p-Caspase-3, molecular mass: 35 kDa); cleaved caspase-3 (c-Caspase-3; molecular mass: 19 and 17 kDa). Poly (ADP-ribose) polymerase (PARP; molecular mass: 116 kDa; cleaved PARP; molecular mass: 89 kDa). As membranes for Nrf2/Egr1 (D) were stripped and reprobed with anti-HO-1/-GCLc antibodies (E), the same β-actin blot is shown.

Techniques Used: Activation Assay, Positive Control, Expressing, Western Blot, SDS Page

15d-PGJ 2 promotes Akt phosphorylation via p38 MAPK activation. (A) MG-63 cells were treated with 15d-PGJ 2 (20 μM) for indicated time periods to follow Akt phosphorylation (pAkt, T 308 ) using Western blot analysis. (B) Cells were incubated with PD169316 (25 μM), LY294002 (10 μM) or Akt-I (5 μM) for 30 min prior to 15d-PGJ 2 treatment (20 μM) for 1 h to follow pAkt expression. For Western blot analysis total protein lysates were subjected to SDS–PAGE. Total Akt expression was used as loading control. One representative blot (A/B [upper panel]) out of three is shown. Densitometric evaluation of immunoreactive bands is given below (A/B [lower panel]). Values are expressed as mean ± SEM ( n = 3). * p ⩽ 0.05 vs. control; # p ⩽ 0.05 vs. 15d-PGJ 2 .
Figure Legend Snippet: 15d-PGJ 2 promotes Akt phosphorylation via p38 MAPK activation. (A) MG-63 cells were treated with 15d-PGJ 2 (20 μM) for indicated time periods to follow Akt phosphorylation (pAkt, T 308 ) using Western blot analysis. (B) Cells were incubated with PD169316 (25 μM), LY294002 (10 μM) or Akt-I (5 μM) for 30 min prior to 15d-PGJ 2 treatment (20 μM) for 1 h to follow pAkt expression. For Western blot analysis total protein lysates were subjected to SDS–PAGE. Total Akt expression was used as loading control. One representative blot (A/B [upper panel]) out of three is shown. Densitometric evaluation of immunoreactive bands is given below (A/B [lower panel]). Values are expressed as mean ± SEM ( n = 3). * p ⩽ 0.05 vs. control; # p ⩽ 0.05 vs. 15d-PGJ 2 .

Techniques Used: Activation Assay, Western Blot, Incubation, Expressing, SDS Page

6) Product Images from "Thymoquinone reduces spinal cord injury by inhibiting inflammatory response, oxidative stress and apoptosis via PPAR-γ and PI3K/Akt pathways"

Article Title: Thymoquinone reduces spinal cord injury by inhibiting inflammatory response, oxidative stress and apoptosis via PPAR-γ and PI3K/Akt pathways

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2018.6072

Effect of thymoquinone on PI3K/Akt protein expression in SCI rats. (A and B) Relative PI3K and p-Akt/Akt protein expression was analyzed by (C) western blotting in SCI rats. ## P
Figure Legend Snippet: Effect of thymoquinone on PI3K/Akt protein expression in SCI rats. (A and B) Relative PI3K and p-Akt/Akt protein expression was analyzed by (C) western blotting in SCI rats. ## P

Techniques Used: Expressing, Western Blot

7) Product Images from "Lewis Y regulates signaling molecules of the transforming growth factor ? pathway in ovarian carcinoma-derived RMG-I cells"

Article Title: Lewis Y regulates signaling molecules of the transforming growth factor ? pathway in ovarian carcinoma-derived RMG-I cells

Journal: International Journal of Oncology

doi: 10.3892/ijo.2011.1296

The effect of LeY antibody on the expression of p-ERK, p-Smad2/3 and p-Akt. (A) A Western blot analysis was performed to measure changes in the phosphorylation levels of ERK, Smad2/3 and Akt at different time-points a fter antibody blocking. (B) Relative intensity of p-ERK, p-Smad2/3 and p-Akt protein levels were expressed as means in bar graphs. Significant differences from control cells were noted as * p
Figure Legend Snippet: The effect of LeY antibody on the expression of p-ERK, p-Smad2/3 and p-Akt. (A) A Western blot analysis was performed to measure changes in the phosphorylation levels of ERK, Smad2/3 and Akt at different time-points a fter antibody blocking. (B) Relative intensity of p-ERK, p-Smad2/3 and p-Akt protein levels were expressed as means in bar graphs. Significant differences from control cells were noted as * p

Techniques Used: Expressing, Western Blot, Blocking Assay

TGF-β1 activates ERK/MAPK and PI3K pathways in RMG-I-H cells. (A) Western blot analysis was performed to detect the expression of p-ERK, ERK, p-Akt and Akt in RMG-I-H cells at different time-points after TGF-β1 stimulation. (B) Relative intensity of p-ERK, ERK, p-Akt and Akt protein were expressed as means in bar graphs, significant differences from control cells were noted as * p
Figure Legend Snippet: TGF-β1 activates ERK/MAPK and PI3K pathways in RMG-I-H cells. (A) Western blot analysis was performed to detect the expression of p-ERK, ERK, p-Akt and Akt in RMG-I-H cells at different time-points after TGF-β1 stimulation. (B) Relative intensity of p-ERK, ERK, p-Akt and Akt protein were expressed as means in bar graphs, significant differences from control cells were noted as * p

Techniques Used: Western Blot, Expressing

Changes in expression levels of elements of ERK and PI3K signaling pathways. (A) Changes of the expression and phosphorylation levels of MEK, ERK, Smad2/3 and Akt in RMG-I, RMG-I-C and RMG-I-H cells. (B) Relative intensity of elements of ERK and PI3K signaling pathways protein levels were expressed as means in bar graphs. Significant differences from RMG-I and RMG-I-C cells were noted as * p
Figure Legend Snippet: Changes in expression levels of elements of ERK and PI3K signaling pathways. (A) Changes of the expression and phosphorylation levels of MEK, ERK, Smad2/3 and Akt in RMG-I, RMG-I-C and RMG-I-H cells. (B) Relative intensity of elements of ERK and PI3K signaling pathways protein levels were expressed as means in bar graphs. Significant differences from RMG-I and RMG-I-C cells were noted as * p

Techniques Used: Expressing

8) Product Images from "TROP2 is epigenetically inactivated and modulates IGF-1R signalling in lung adenocarcinoma"

Article Title: TROP2 is epigenetically inactivated and modulates IGF-1R signalling in lung adenocarcinoma

Journal: EMBO Molecular Medicine

doi: 10.1002/emmm.201200222

Trop-2 can bind to IGF-1 and interfere with IGF-1R signalling BLAST analysis of Trop-2 against the NCBI database ( http://www.ncbi.nlm.nih.gov/ ) shows that the members of the TROP and insulin growth factor binding protein (IGFBP) families share a conserved thyroglobulin type-1 domain. Black and purple boxes indicated the glycosylation and phosphorylation sites, respectively. Vectors encoding TROP2 and mIGF-1 were co-transfected into H1299 lung CL, and the interaction of TROP2 and mIGF-1 was examined by co-IP. WB, western blot. Overexpression of TROP2 in H1299 cells reduced the activation of IGF-1R signalling, while TROP2 knockdown activated IGF-1R signalling in H322M cells. Forced expression of TROP2 in H1299 cells suppressed the IGF-1-induced phosphorylation of AKT. The expression of downstream mediators of IGF-1R signalling ( e.g. β-catenin) decreased when TROP2 was overexpressed in H1299 and H23 cells, and increased following TROP2 knockdown in H322M and PC-9 cells. Slug expression was induced by TROP2 knockdown.
Figure Legend Snippet: Trop-2 can bind to IGF-1 and interfere with IGF-1R signalling BLAST analysis of Trop-2 against the NCBI database ( http://www.ncbi.nlm.nih.gov/ ) shows that the members of the TROP and insulin growth factor binding protein (IGFBP) families share a conserved thyroglobulin type-1 domain. Black and purple boxes indicated the glycosylation and phosphorylation sites, respectively. Vectors encoding TROP2 and mIGF-1 were co-transfected into H1299 lung CL, and the interaction of TROP2 and mIGF-1 was examined by co-IP. WB, western blot. Overexpression of TROP2 in H1299 cells reduced the activation of IGF-1R signalling, while TROP2 knockdown activated IGF-1R signalling in H322M cells. Forced expression of TROP2 in H1299 cells suppressed the IGF-1-induced phosphorylation of AKT. The expression of downstream mediators of IGF-1R signalling ( e.g. β-catenin) decreased when TROP2 was overexpressed in H1299 and H23 cells, and increased following TROP2 knockdown in H322M and PC-9 cells. Slug expression was induced by TROP2 knockdown.

Techniques Used: Binding Assay, Transfection, Co-Immunoprecipitation Assay, Western Blot, Over Expression, Activation Assay, Expressing

TROP2 expression modulates cell proliferation and colony formation Trop-2 was overexpressed in H1299 cells and knocked down in H322M cells, and the activities of AKT and ERK were determined by Western blotting with anti-pAKT and anti-p-ERK antibodies, respectively. Forced expression of Trop-2 in H1299 cells inhibited cell proliferation compared to the vector control, as determined by cell counting. Conversely, Trop-2 knockdown by either lenti-shTROP2A or lenti-shTROP2C enhanced cell proliferation. The tumour growth ability of lung CL was measured by the formation of colonies in soft agar. The results are presented as means ± SD, data were compared between groups using the t -test, and p
Figure Legend Snippet: TROP2 expression modulates cell proliferation and colony formation Trop-2 was overexpressed in H1299 cells and knocked down in H322M cells, and the activities of AKT and ERK were determined by Western blotting with anti-pAKT and anti-p-ERK antibodies, respectively. Forced expression of Trop-2 in H1299 cells inhibited cell proliferation compared to the vector control, as determined by cell counting. Conversely, Trop-2 knockdown by either lenti-shTROP2A or lenti-shTROP2C enhanced cell proliferation. The tumour growth ability of lung CL was measured by the formation of colonies in soft agar. The results are presented as means ± SD, data were compared between groups using the t -test, and p

Techniques Used: Expressing, Western Blot, Plasmid Preparation, Cell Counting

9) Product Images from "High sensitivity isoelectric focusing to establish a signaling biomarker for the diagnosis of human colorectal cancer"

Article Title: High sensitivity isoelectric focusing to establish a signaling biomarker for the diagnosis of human colorectal cancer

Journal: BMC Cancer

doi: 10.1186/s12885-016-2725-z

Detection of total AKT protein and phospho-protein by isoelectric focusing. Plots ( d-f, h ) show values after normalization to HSP70 levels analyzed in parallel in each sample. Symbols in plots: Red; KRAS mutated, green; BRAF mutated, blue; wild type (WT) with regard to KRAS and BRAF , black; unclear for KRAS and BRAF . a. Immunoblotting of selected tissue samples with antibodies against pAKT (AKT pS473) and total AKT protein. Blotting for β2 microglobulin (β2M) was used as a loading control. b. Representative electropherogram showing phosphorylated and non-phosphorylated AKT peaks. Blue and green lines indicate electropherograms of samples digested (green) or not (blue) with lambda phosphatase. Inset; electropherogram showing HSP70 run in parallel. c. Immunoblotting of HUVEC (±VEGF for 15 min) cell lysate with antibodies against pAKT and total AKT protein. Blotting for tubulin was used as to monitor equal loading. Control; without any incubation; Buffer; control lysate incubated with buffer, Phosphatase; lysate incubated with lambda phosphatase enzyme. d. Plot of total AKT peak areas (P1–P8) in the different samples. e. Plot of pAKT peak areas (present before but not after lambda phosphatase treatment; P1–P4 and P6). f. Plot of the ratio of pAKT/AKT peak areas. g. Representative electropherogram of p70S6 kinase expression. h. Plot of p70S6 kinase expression normalized to HSP70
Figure Legend Snippet: Detection of total AKT protein and phospho-protein by isoelectric focusing. Plots ( d-f, h ) show values after normalization to HSP70 levels analyzed in parallel in each sample. Symbols in plots: Red; KRAS mutated, green; BRAF mutated, blue; wild type (WT) with regard to KRAS and BRAF , black; unclear for KRAS and BRAF . a. Immunoblotting of selected tissue samples with antibodies against pAKT (AKT pS473) and total AKT protein. Blotting for β2 microglobulin (β2M) was used as a loading control. b. Representative electropherogram showing phosphorylated and non-phosphorylated AKT peaks. Blue and green lines indicate electropherograms of samples digested (green) or not (blue) with lambda phosphatase. Inset; electropherogram showing HSP70 run in parallel. c. Immunoblotting of HUVEC (±VEGF for 15 min) cell lysate with antibodies against pAKT and total AKT protein. Blotting for tubulin was used as to monitor equal loading. Control; without any incubation; Buffer; control lysate incubated with buffer, Phosphatase; lysate incubated with lambda phosphatase enzyme. d. Plot of total AKT peak areas (P1–P8) in the different samples. e. Plot of pAKT peak areas (present before but not after lambda phosphatase treatment; P1–P4 and P6). f. Plot of the ratio of pAKT/AKT peak areas. g. Representative electropherogram of p70S6 kinase expression. h. Plot of p70S6 kinase expression normalized to HSP70

Techniques Used: Incubation, Expressing

10) Product Images from "Pressure Combined with Ischemia/Reperfusion Injury Induces Deep Tissue Injury via Endoplasmic Reticulum Stress in a Rat Pressure Ulcer Model"

Article Title: Pressure Combined with Ischemia/Reperfusion Injury Induces Deep Tissue Injury via Endoplasmic Reticulum Stress in a Rat Pressure Ulcer Model

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms17030284

Phospho-Akt and phospho-GSK3β were downregulated in compressed tissues. The protein level of phospho-Akt (p-Akt), Akt, phospho-GSK3β (p-GSK3β), and GSK3β were determined by western blotting. ( A ) GAPDH was used as a protein loading control and for band density normalization; ( B , C ) Bar diagram of p-Akt/Akt and p-GSK3β/GSK3β ratios was calculated and plotted. Data are represented as the mean values ± SD. n = 8. * p
Figure Legend Snippet: Phospho-Akt and phospho-GSK3β were downregulated in compressed tissues. The protein level of phospho-Akt (p-Akt), Akt, phospho-GSK3β (p-GSK3β), and GSK3β were determined by western blotting. ( A ) GAPDH was used as a protein loading control and for band density normalization; ( B , C ) Bar diagram of p-Akt/Akt and p-GSK3β/GSK3β ratios was calculated and plotted. Data are represented as the mean values ± SD. n = 8. * p

Techniques Used: Western Blot

11) Product Images from "Role of SIRT1-mediated mitochondrial and Akt pathways in glioblastoma cell death induced by Cotinus coggygria flavonoid nanoliposomes"

Article Title: Role of SIRT1-mediated mitochondrial and Akt pathways in glioblastoma cell death induced by Cotinus coggygria flavonoid nanoliposomes

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S82282

CCF-NLs induced cell death was associated with SIRT1/p53-mediated mitochondrial and Akt pathway in glioblastoma DBTRG-05MG cells. Abbreviations: CCF-NLs, Cotinus coggygria flavonoid nanoliposomes; Bcl-2, B-cell lymphoma/leukemia 2; Bax, Bcl-2-associated X protein; Cyto.c, cytochrome c.
Figure Legend Snippet: CCF-NLs induced cell death was associated with SIRT1/p53-mediated mitochondrial and Akt pathway in glioblastoma DBTRG-05MG cells. Abbreviations: CCF-NLs, Cotinus coggygria flavonoid nanoliposomes; Bcl-2, B-cell lymphoma/leukemia 2; Bax, Bcl-2-associated X protein; Cyto.c, cytochrome c.

Techniques Used:

12) Product Images from "Safflower yellow B suppresses HepG2 cell injury induced by oxidative stress through the AKT/Nrf2 pathway"

Article Title: Safflower yellow B suppresses HepG2 cell injury induced by oxidative stress through the AKT/Nrf2 pathway

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2016.2462

Role of AKT in safflower yellow B (SYB)-induced nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) upregulation. (A) Effects of SYB (50, 100 and 150 nmol/l) on AKT protein expression in HepG2 cells exposed to H 2 O 2 (200 µ mol/l). (B) Role of AKT in SYB-induced Nrf2 expression. Cells were pre-treated with SYB (150 nmol/l) or LY294002 (10 µ M) for 24 h, then exposed to H 2 O 2 (200 µ mol/l). Total cell lysates were prepared and subjected to western blot analysis. β-actin was used as a loading control. (C) GSH-Px and (D) superoxide dismutase (SOD) levels were measured using respective kits. Data represent the means ± SD. ## P
Figure Legend Snippet: Role of AKT in safflower yellow B (SYB)-induced nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) upregulation. (A) Effects of SYB (50, 100 and 150 nmol/l) on AKT protein expression in HepG2 cells exposed to H 2 O 2 (200 µ mol/l). (B) Role of AKT in SYB-induced Nrf2 expression. Cells were pre-treated with SYB (150 nmol/l) or LY294002 (10 µ M) for 24 h, then exposed to H 2 O 2 (200 µ mol/l). Total cell lysates were prepared and subjected to western blot analysis. β-actin was used as a loading control. (C) GSH-Px and (D) superoxide dismutase (SOD) levels were measured using respective kits. Data represent the means ± SD. ## P

Techniques Used: Expressing, Western Blot

13) Product Images from "Tanshinone IIA inhibits angiogenesis in human endothelial progenitor cells in vitro and in vivo"

Article Title: Tanshinone IIA inhibits angiogenesis in human endothelial progenitor cells in vitro and in vivo

Journal: Oncotarget

doi: 10.18632/oncotarget.22649

Schema of tanshinone IIA-induced anti-angiogenesis property in human endothelial progenitor cells Tanshinone IIA reduces EPCs angiogenesis in vitro and in vivo by controlling the PLC, Akt and JNK signaling pathways.
Figure Legend Snippet: Schema of tanshinone IIA-induced anti-angiogenesis property in human endothelial progenitor cells Tanshinone IIA reduces EPCs angiogenesis in vitro and in vivo by controlling the PLC, Akt and JNK signaling pathways.

Techniques Used: In Vitro, In Vivo, Planar Chromatography

Effects of tanshinone IIA on PLC and Akt signaling pathways in human EPCs EPCs were incubated with VEGF-A (100 ng/ml) and the indicated concentrations of tanshinone IIA for 24 h. Phosphorylation of PLC (A) and Akt (B) was examined by Western blotting. Data represent the mean ± S.E.M. of four independent experiments. * , p
Figure Legend Snippet: Effects of tanshinone IIA on PLC and Akt signaling pathways in human EPCs EPCs were incubated with VEGF-A (100 ng/ml) and the indicated concentrations of tanshinone IIA for 24 h. Phosphorylation of PLC (A) and Akt (B) was examined by Western blotting. Data represent the mean ± S.E.M. of four independent experiments. * , p

Techniques Used: Planar Chromatography, Incubation, Western Blot

14) Product Images from "Mapping Pathological Phenotypes in a Mouse Model of CDKL5 Disorder"

Article Title: Mapping Pathological Phenotypes in a Mouse Model of CDKL5 Disorder

Journal: PLoS ONE

doi: 10.1371/journal.pone.0091613

Defective cellular signalling in Cdkl5 knockout mice. ( A ) Western blot analysis of whole brain protein extracts from adult female and male wild-type and Cdkl5 mutant mice. Tubulin was included as a loading control. No change was seen in MeCP2 (top panel) and BDNF (lower panel) levels in mutant Cdkl5 mice compared with wild-type controls (X/X, N = 10; -/X, N = 6; -/-, N = 10; X/Y, N = 5; -/Y, N = 6). ( B ) Western blot analysis of hyppocampal protein extracts from P19 female and male wild-type and Cdkl5 mutant mice. Decreased pAKT was observed in Cdkl5 mutant mic4 compared with wild-type controls (X/X, N = 4; -/X, N = 6/8; -/-, N = 6; X/Y, N = 3; -/Y, N = 5. ( C ) Western blot quantification revealed a significant decrease in phospho-Akt immunoreactivity protein in mutant Cdkl5 mice compared with wild-type controls (female: WT, N = 10, HET, N = 6, KO, N = 10; male: WT, N = 5, KO, N = 6; mean ± SEM) (mean ± SEM; *P
Figure Legend Snippet: Defective cellular signalling in Cdkl5 knockout mice. ( A ) Western blot analysis of whole brain protein extracts from adult female and male wild-type and Cdkl5 mutant mice. Tubulin was included as a loading control. No change was seen in MeCP2 (top panel) and BDNF (lower panel) levels in mutant Cdkl5 mice compared with wild-type controls (X/X, N = 10; -/X, N = 6; -/-, N = 10; X/Y, N = 5; -/Y, N = 6). ( B ) Western blot analysis of hyppocampal protein extracts from P19 female and male wild-type and Cdkl5 mutant mice. Decreased pAKT was observed in Cdkl5 mutant mic4 compared with wild-type controls (X/X, N = 4; -/X, N = 6/8; -/-, N = 6; X/Y, N = 3; -/Y, N = 5. ( C ) Western blot quantification revealed a significant decrease in phospho-Akt immunoreactivity protein in mutant Cdkl5 mice compared with wild-type controls (female: WT, N = 10, HET, N = 6, KO, N = 10; male: WT, N = 5, KO, N = 6; mean ± SEM) (mean ± SEM; *P

Techniques Used: Knock-Out, Mouse Assay, Western Blot, Mutagenesis

15) Product Images from "Soya-cerebroside, an extract of Cordyceps militaris, suppresses monocyte migration and prevents cartilage degradation in inflammatory animal models"

Article Title: Soya-cerebroside, an extract of Cordyceps militaris, suppresses monocyte migration and prevents cartilage degradation in inflammatory animal models

Journal: Scientific Reports

doi: 10.1038/srep43205

AMPK/AKT signaling pathways are involved in soya-cerebroside mediated miR-432 expression. ( A ) OASFs were treated with various soya-cerebroside concentrations in the presence or absence of IL-1β for 1 h, cells were collected, and AMPK and AKT phosphorylation was examined by Western blot. ( B ) OASFs were pretreated with Ara A, Compound C, and AKTi for 30 min followed by stimulation with IL-1β for 24 h; miR-432 expression was examined by qPCR. ( C ) OASFs were pretreated with Ara A, Compound C, and AKTi for 30 min, followed by stimulation with IL-1β for 24 h. The wt-SP1-3′-UTR relative luciferase/renilla activities were measured as described in the Methods section. ( D ) OASFs were treated as explained in C, monocyte migration was examined by in vitro chemotaxis assay *p
Figure Legend Snippet: AMPK/AKT signaling pathways are involved in soya-cerebroside mediated miR-432 expression. ( A ) OASFs were treated with various soya-cerebroside concentrations in the presence or absence of IL-1β for 1 h, cells were collected, and AMPK and AKT phosphorylation was examined by Western blot. ( B ) OASFs were pretreated with Ara A, Compound C, and AKTi for 30 min followed by stimulation with IL-1β for 24 h; miR-432 expression was examined by qPCR. ( C ) OASFs were pretreated with Ara A, Compound C, and AKTi for 30 min, followed by stimulation with IL-1β for 24 h. The wt-SP1-3′-UTR relative luciferase/renilla activities were measured as described in the Methods section. ( D ) OASFs were treated as explained in C, monocyte migration was examined by in vitro chemotaxis assay *p

Techniques Used: Expressing, Western Blot, Acetylene Reduction Assay, Real-time Polymerase Chain Reaction, Luciferase, Migration, In Vitro, Chemotaxis Assay

16) Product Images from "Trigonostemon reidioides modulates endothelial cell proliferation, migration and tube formation via downregulation of the Akt signaling pathway"

Article Title: Trigonostemon reidioides modulates endothelial cell proliferation, migration and tube formation via downregulation of the Akt signaling pathway

Journal: Oncology Letters

doi: 10.3892/ol.2017.6760

ETR inhibits mitogen-induced Akt and MMP-2 activities. Quiescent cells were treated with ETR (1, 10 and 25 µg/ml) for 15 min. (A) Cell lysates were analyzed by western blotting with anti-p-ERK, anti-ERK, anti-p-Akt, anti-Akt, and anti-p-p70S6K antibodies. (B) Gelatin zymogram analysis was performed using basic EBM 2 medium from cell culture. Zymogram gel loading was normalized to total protein concentration. Results are representative of ≥3 independent experiments. ETR, ethanolic extract of Trigonostemon reidioides ; CM, complete medium; MMP, matrix metalloproteinase; p, phosphorylated; ERK, extracellular signal-regulated kinase; p70S6K, p70 ribosomal S6 kinase.
Figure Legend Snippet: ETR inhibits mitogen-induced Akt and MMP-2 activities. Quiescent cells were treated with ETR (1, 10 and 25 µg/ml) for 15 min. (A) Cell lysates were analyzed by western blotting with anti-p-ERK, anti-ERK, anti-p-Akt, anti-Akt, and anti-p-p70S6K antibodies. (B) Gelatin zymogram analysis was performed using basic EBM 2 medium from cell culture. Zymogram gel loading was normalized to total protein concentration. Results are representative of ≥3 independent experiments. ETR, ethanolic extract of Trigonostemon reidioides ; CM, complete medium; MMP, matrix metalloproteinase; p, phosphorylated; ERK, extracellular signal-regulated kinase; p70S6K, p70 ribosomal S6 kinase.

Techniques Used: Western Blot, Cell Culture, Protein Concentration

17) Product Images from "Degradation of Epidermal Growth Factor Receptor Mediates Dasatinib-Induced Apoptosis in Head and Neck Squamous Cell Carcinoma Cells 1Degradation of Epidermal Growth Factor Receptor Mediates Dasatinib-Induced Apoptosis in Head and Neck Squamous Cell Carcinoma Cells 1 2"

Article Title: Degradation of Epidermal Growth Factor Receptor Mediates Dasatinib-Induced Apoptosis in Head and Neck Squamous Cell Carcinoma Cells 1Degradation of Epidermal Growth Factor Receptor Mediates Dasatinib-Induced Apoptosis in Head and Neck Squamous Cell Carcinoma Cells 1 2

Journal: Neoplasia (New York, N.Y.)

doi:

Down-regulation of EGFR by dasatinib correlates with the sensitivity of dasatinib. (A, B) Expression of EGFR and downstream Akt and Erk in dasatinib-sensitive (A) and -resistant (B) cells treated with dasatinib 1 µ M for indicated intervals. Upper
Figure Legend Snippet: Down-regulation of EGFR by dasatinib correlates with the sensitivity of dasatinib. (A, B) Expression of EGFR and downstream Akt and Erk in dasatinib-sensitive (A) and -resistant (B) cells treated with dasatinib 1 µ M for indicated intervals. Upper

Techniques Used: Expressing

Role of EGFR in dasatinib-induced apoptosis. (A, B) Effect of EGF addition to dasatinib (1 µ M)-induced apoptosis and inactivation of Akt and Erk in sensitive HSC3 (A) and Ca9-22 (B) cells. Cells were treated with dasatinib (1 µ M, 0 hour)
Figure Legend Snippet: Role of EGFR in dasatinib-induced apoptosis. (A, B) Effect of EGF addition to dasatinib (1 µ M)-induced apoptosis and inactivation of Akt and Erk in sensitive HSC3 (A) and Ca9-22 (B) cells. Cells were treated with dasatinib (1 µ M, 0 hour)

Techniques Used:

18) Product Images from "Apigenin attenuates insulin-like growth factor-I signaling in an autochthonous mouse prostate cancer model"

Article Title: Apigenin attenuates insulin-like growth factor-I signaling in an autochthonous mouse prostate cancer model

Journal: Pharmaceutical research

doi: 10.1007/s11095-011-0625-0

Levels of insulin like growth factor-I(IGF-I), insulin like growth factor binding protein-3 (IGFBP-3), phosphorylated forms of Akt and extracellular signal-regulated kinases1/2 (ERK1/2), vascular endothelial growth factor (VEGF) and urokinase plasminogen activator (uPA) in the serum and dorso-lateral prostate during progressive stages of prostate cancer development in TRAMP mice and age-matched non-transgenic littermates. Serum levels of IGF-I, IGFBP-3, VEGF and uPA; whereas phosphorylated levels of Akt (Ser473) and ERK1 (T202/Y204)/ ERK2 (T185/Y187) were detected in the tissue lysates obtained from the dorso-lateral prostates by Enzyme-linked immunosorbant assay (ELISA) at 8, 16, 24 and 32 weeks of age in non-transgenic and TRAMP mice. Data represents the mean ± SE of 6 mice. * P
Figure Legend Snippet: Levels of insulin like growth factor-I(IGF-I), insulin like growth factor binding protein-3 (IGFBP-3), phosphorylated forms of Akt and extracellular signal-regulated kinases1/2 (ERK1/2), vascular endothelial growth factor (VEGF) and urokinase plasminogen activator (uPA) in the serum and dorso-lateral prostate during progressive stages of prostate cancer development in TRAMP mice and age-matched non-transgenic littermates. Serum levels of IGF-I, IGFBP-3, VEGF and uPA; whereas phosphorylated levels of Akt (Ser473) and ERK1 (T202/Y204)/ ERK2 (T185/Y187) were detected in the tissue lysates obtained from the dorso-lateral prostates by Enzyme-linked immunosorbant assay (ELISA) at 8, 16, 24 and 32 weeks of age in non-transgenic and TRAMP mice. Data represents the mean ± SE of 6 mice. * P

Techniques Used: Binding Assay, Mouse Assay, Transgenic Assay, Enzyme-linked Immunosorbent Assay

19) Product Images from "Mevastatin ameliorates sphingosine 1‐phosphate‐induced COX‐2/PGE2‐dependent cell migration via FoxO1 and CREB phosphorylation and translocation) Mevastatin ameliorates sphingosine 1‐phosphate‐induced COX‐2/PGE2‐dependent cell migration via FoxO1 and CREB phosphorylation and translocation"

Article Title: Mevastatin ameliorates sphingosine 1‐phosphate‐induced COX‐2/PGE2‐dependent cell migration via FoxO1 and CREB phosphorylation and translocation) Mevastatin ameliorates sphingosine 1‐phosphate‐induced COX‐2/PGE2‐dependent cell migration via FoxO1 and CREB phosphorylation and translocation

Journal: British Journal of Pharmacology

doi: 10.1111/bph.13326

Schematic diagram illustrating the proposed signalling pathways involved in S1P‐induced COX‐2 expression and cell migration in HTSMCs. S1P induces COX‐2/PGE 2 ‐depen dent cell migration via Nox2/ROS‐ and PI3K/Akt‐dependent
Figure Legend Snippet: Schematic diagram illustrating the proposed signalling pathways involved in S1P‐induced COX‐2 expression and cell migration in HTSMCs. S1P induces COX‐2/PGE 2 ‐depen dent cell migration via Nox2/ROS‐ and PI3K/Akt‐dependent

Techniques Used: Expressing, Migration

20) Product Images from "Expression of HNF4G and its potential functions in lung cancer"

Article Title: Expression of HNF4G and its potential functions in lung cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.22933

Involvement of AKT pathway in the functions of HNF4G A549 cells were infected with control virus (Vector) or HNF4G virus, and the protein levels of p-AKT, AKT and HNF4G ( A ), cell proliferation ( B ), cell cycle distribution ( C ) and cell apoptosis ( D ) were determined in the present of either DMSO or AKT inhibitor (MK-2206, 2 μM). The experiments were repeated three times and representative images were shown. * P
Figure Legend Snippet: Involvement of AKT pathway in the functions of HNF4G A549 cells were infected with control virus (Vector) or HNF4G virus, and the protein levels of p-AKT, AKT and HNF4G ( A ), cell proliferation ( B ), cell cycle distribution ( C ) and cell apoptosis ( D ) were determined in the present of either DMSO or AKT inhibitor (MK-2206, 2 μM). The experiments were repeated three times and representative images were shown. * P

Techniques Used: Infection, Plasmid Preparation

21) Product Images from "The Hexane Fraction of Bursera microphylla A. Gray Induces p21-Mediated Anti-Proliferative and Pro-Apoptotic Effects in Human Cancer-Derived Cell Lines"

Article Title: The Hexane Fraction of Bursera microphylla A. Gray Induces p21-Mediated Anti-Proliferative and Pro-Apoptotic Effects in Human Cancer-Derived Cell Lines

Journal: Integrative Cancer Therapies

doi: 10.1177/1534735417696721

Time-course effects of BM-H on MAPK pathway. Western blot bands represent phosphorylated ERK (p-ERK), total ERK (ERK), actin (as a housekeeping gene for ERK), phosphorylated p38 (p-p38), total p38 (p38), phosphorylated Akt (p-Akt), total Akt (Akt), and the housekeeping gene tubulin (to control both p-38 and Akt). Proteins were extracted from OCI cells treated with the vehicle (control) or with BM-H (BM-H) after 4, 8, 14, or 24 hours. Western blots are representative of 3 independent experiments.
Figure Legend Snippet: Time-course effects of BM-H on MAPK pathway. Western blot bands represent phosphorylated ERK (p-ERK), total ERK (ERK), actin (as a housekeeping gene for ERK), phosphorylated p38 (p-p38), total p38 (p38), phosphorylated Akt (p-Akt), total Akt (Akt), and the housekeeping gene tubulin (to control both p-38 and Akt). Proteins were extracted from OCI cells treated with the vehicle (control) or with BM-H (BM-H) after 4, 8, 14, or 24 hours. Western blots are representative of 3 independent experiments.

Techniques Used: Western Blot

22) Product Images from "The histone acetyltransferases CBP/p300 are degraded in NIH 3T3 cells by activation of Ras signalling pathway"

Article Title: The histone acetyltransferases CBP/p300 are degraded in NIH 3T3 cells by activation of Ras signalling pathway

Journal: Biochemical Journal

doi: 10.1042/BJ20060052

Ras V12-expressing cell lines display transforming activity ( A ) Cell doubling times of NIH 3T3 clones overexpressing different H-, K- or N-Ras V12 mutants, or transfected only with the vector, were measured as described in the Materials and methods section. Doubling time was determined by counting cells every 2 days. Results are means±S.D. for three independent experiments where each sample was analysed in triplicate. ( B ) The expression levels of AU5–Ras V12 proteins of the above NIH 3T3 clones were detected by immunoblotting (IB) with monoclonal anti-AU5 (top panel); cell extracts from NIH 3T3 fibroblasts transfected with the vector pCEFL-KZ-AU5 (control, C) were included as a negative control. All cells were serum-starved for 18 h and then the ERK (p42 and p44 proteins) and Akt phosphorylation levels were determined using specific anti-phospho- and full antibodies. Ral A-GTP was recovered from cell lysates by binding to immobilized GST–Ral-BD and detected by immunoblotting with the corresponding anti-(Ral A) monoclonal antibody. The expression levels of the endogenous Ral A protein were detected by immunoblotting of the cell extracts with the corresponding anti-(Ral A) monoclonal antibody. Results are from a representative of four separate experiments.
Figure Legend Snippet: Ras V12-expressing cell lines display transforming activity ( A ) Cell doubling times of NIH 3T3 clones overexpressing different H-, K- or N-Ras V12 mutants, or transfected only with the vector, were measured as described in the Materials and methods section. Doubling time was determined by counting cells every 2 days. Results are means±S.D. for three independent experiments where each sample was analysed in triplicate. ( B ) The expression levels of AU5–Ras V12 proteins of the above NIH 3T3 clones were detected by immunoblotting (IB) with monoclonal anti-AU5 (top panel); cell extracts from NIH 3T3 fibroblasts transfected with the vector pCEFL-KZ-AU5 (control, C) were included as a negative control. All cells were serum-starved for 18 h and then the ERK (p42 and p44 proteins) and Akt phosphorylation levels were determined using specific anti-phospho- and full antibodies. Ral A-GTP was recovered from cell lysates by binding to immobilized GST–Ral-BD and detected by immunoblotting with the corresponding anti-(Ral A) monoclonal antibody. The expression levels of the endogenous Ral A protein were detected by immunoblotting of the cell extracts with the corresponding anti-(Ral A) monoclonal antibody. Results are from a representative of four separate experiments.

Techniques Used: Expressing, Activity Assay, Clone Assay, Transfection, Plasmid Preparation, Negative Control, Binding Assay

PDGF stimulation decreases CBP/p300 levels in NIH 3T3 cells NIH 3T3 fibroblasts were stimulated with PDGF (1 mg/ml) for 0, 15 (15′), 30 (30′), 60 (60′) min, 1 day (1d), 2 days (2d) or 3 days (3d). Total cell extracts cells were prepared, the total protein amount was adjusted and analysed as ( A ) and ( B ). ( A ) The ERK (p42 and p44 proteins) and Akt phosphorylation levels were determined using specific anti-phospho- and full antibodies. The fold increase values of p-ERK and p-Akt are the means for three separate assays (in each case with a S.D. lower than 15% of the average). ( B ) The CBP and p300 levels were monitored by immunoblot analysis using antibodies against CBP, p300 and tubulin (as an internal control). The diagrams shown relative levels of CBP and p300 after PDGF stimulation of NIH 3T3 cells. Results are means±S.D. for three independent assays. IB, immunoblot.
Figure Legend Snippet: PDGF stimulation decreases CBP/p300 levels in NIH 3T3 cells NIH 3T3 fibroblasts were stimulated with PDGF (1 mg/ml) for 0, 15 (15′), 30 (30′), 60 (60′) min, 1 day (1d), 2 days (2d) or 3 days (3d). Total cell extracts cells were prepared, the total protein amount was adjusted and analysed as ( A ) and ( B ). ( A ) The ERK (p42 and p44 proteins) and Akt phosphorylation levels were determined using specific anti-phospho- and full antibodies. The fold increase values of p-ERK and p-Akt are the means for three separate assays (in each case with a S.D. lower than 15% of the average). ( B ) The CBP and p300 levels were monitored by immunoblot analysis using antibodies against CBP, p300 and tubulin (as an internal control). The diagrams shown relative levels of CBP and p300 after PDGF stimulation of NIH 3T3 cells. Results are means±S.D. for three independent assays. IB, immunoblot.

Techniques Used:

23) Product Images from "TCR-Induced Akt Serine 473 Phosphorylation Is Regulated by Protein Kinase C-Alpha"

Article Title: TCR-Induced Akt Serine 473 Phosphorylation Is Regulated by Protein Kinase C-Alpha

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2010.07.126

Knockdown of PKC-α in Jurkat cells decreases TCR-induced Akt phosphorylation. Jurkat cells were transfected with PKC-α or nonsense siRNA as described in Methods. Forty-eight hours later, the transfected cells were starved in RPMI 1640
Figure Legend Snippet: Knockdown of PKC-α in Jurkat cells decreases TCR-induced Akt phosphorylation. Jurkat cells were transfected with PKC-α or nonsense siRNA as described in Methods. Forty-eight hours later, the transfected cells were starved in RPMI 1640

Techniques Used: Transfection

PKC-α is the PDK-2 responsible for phosphorylating Akt at Ser 473 upon TCR stimulation. T cells isolated from B6 mice were stimulated with anti-CD3 and lysed. PKC-α (A) or PKC-β (B) were immunoprecipitated and in vitro kinase assays
Figure Legend Snippet: PKC-α is the PDK-2 responsible for phosphorylating Akt at Ser 473 upon TCR stimulation. T cells isolated from B6 mice were stimulated with anti-CD3 and lysed. PKC-α (A) or PKC-β (B) were immunoprecipitated and in vitro kinase assays

Techniques Used: Isolation, Mouse Assay, Immunoprecipitation, In Vitro

Conventional PKC positively regulates TCR-induced phosphorylation of Akt. (A) T cells purified from C57BL/6 (B6) or PKC-θ −/− mice were stimulated with anti-CD3 (1μg/ml) for the indicated time points and lysed in RIPA buffer.
Figure Legend Snippet: Conventional PKC positively regulates TCR-induced phosphorylation of Akt. (A) T cells purified from C57BL/6 (B6) or PKC-θ −/− mice were stimulated with anti-CD3 (1μg/ml) for the indicated time points and lysed in RIPA buffer.

Techniques Used: Purification, Mouse Assay

24) Product Images from "Chitosan oligosaccharides suppress LPS-induced IL-8 expression in human umbilical vein endothelial cells through blockade of p38 and Akt protein kinases"

Article Title: Chitosan oligosaccharides suppress LPS-induced IL-8 expression in human umbilical vein endothelial cells through blockade of p38 and Akt protein kinases

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2011.10

Inhibitory effect of COS on LPS-induced over-expression of phosphorylated p38 MAPK (A) and Akt (B) in HUVECs. Cells were pretreated with COS (50, 100, and 200 μg/mL) for 24 h and then exposed to LPS (100 ng/mL) for 10 min for p38 detection and for 1 h for Akt detection. After treatment, cell lysates were extracted and the protein levels of phosphorylated p38 (p-p38) MAPK, total p38 (t-p38) MAPK, phosphorylated Akt (p-Akt) and total Akt (t-Akt) were determined by Western blot analysis as described in Materials and methods. Data are representative of three experiments (mans±SD). b P
Figure Legend Snippet: Inhibitory effect of COS on LPS-induced over-expression of phosphorylated p38 MAPK (A) and Akt (B) in HUVECs. Cells were pretreated with COS (50, 100, and 200 μg/mL) for 24 h and then exposed to LPS (100 ng/mL) for 10 min for p38 detection and for 1 h for Akt detection. After treatment, cell lysates were extracted and the protein levels of phosphorylated p38 (p-p38) MAPK, total p38 (t-p38) MAPK, phosphorylated Akt (p-Akt) and total Akt (t-Akt) were determined by Western blot analysis as described in Materials and methods. Data are representative of three experiments (mans±SD). b P

Techniques Used: Over Expression, Western Blot

25) Product Images from "Corrective effects of hepatotoxicity by hepatic Dyrk1a gene delivery in mice with intermediate hyperhomocysteinemia"

Article Title: Corrective effects of hepatotoxicity by hepatic Dyrk1a gene delivery in mice with intermediate hyperhomocysteinemia

Journal: Molecular Genetics and Metabolism Reports

doi: 10.1016/j.ymgmr.2014.12.007

Effect of hepatic overexpression of Dyrk1a on phospho-Akt, GSK3, phospho-GSK3, and cyclin D1 levels in mice with intermediate hhcy. Phosphorylation of Akt (a), GSK3 alpha and beta expression (b), phosphorylation of GSK3 alpha and beta on ser 21 and 9 (c), and cyclin D1 expression (d) in the liver of wild-type (Cbs +/+ ) mice and Cbs +/− mice supplemented with methionine and injected (Cbs +/− Met/AdDYRK1A) or uninjected (Cbs +/− Met) with AdDYRK1A. GSK3 and cyclin D1 expression were determined by slot blotting, and values were obtained by normalization of images from GSK3 and cyclin D1 to total proteins colored with Ponceau-S. Relative protein expression was determined by normalization from p-Akt or p-GSK3 with that of total Akt or GSK3. Data were normalized to the mean of wild-type mice (Cbs +/+ ). Data correspond to the medians with interquartile ranges. n = number of mice.
Figure Legend Snippet: Effect of hepatic overexpression of Dyrk1a on phospho-Akt, GSK3, phospho-GSK3, and cyclin D1 levels in mice with intermediate hhcy. Phosphorylation of Akt (a), GSK3 alpha and beta expression (b), phosphorylation of GSK3 alpha and beta on ser 21 and 9 (c), and cyclin D1 expression (d) in the liver of wild-type (Cbs +/+ ) mice and Cbs +/− mice supplemented with methionine and injected (Cbs +/− Met/AdDYRK1A) or uninjected (Cbs +/− Met) with AdDYRK1A. GSK3 and cyclin D1 expression were determined by slot blotting, and values were obtained by normalization of images from GSK3 and cyclin D1 to total proteins colored with Ponceau-S. Relative protein expression was determined by normalization from p-Akt or p-GSK3 with that of total Akt or GSK3. Data were normalized to the mean of wild-type mice (Cbs +/+ ). Data correspond to the medians with interquartile ranges. n = number of mice.

Techniques Used: Over Expression, Mouse Assay, Expressing, Injection

26) Product Images from "MicroRNA-498 promotes proliferation and migration by targeting the tumor suppressor PTEN in breast cancer cells"

Article Title: MicroRNA-498 promotes proliferation and migration by targeting the tumor suppressor PTEN in breast cancer cells

Journal: Carcinogenesis

doi: 10.1093/carcin/bgy092

miR-498 promotes cell proliferation and cell cycle arrest by suppressing PTEN and increasing p-Akt levels in TNBC cells. ( A ) MDA-MB-231 cells were transduced with the indicated expression plasmids and analyzed by immunoblotting with specific antibodies. ( B ) Levels of miR-498 expression were measured by qRT-PCR. ( C ) Similar to A, except that BT-549 (PTEN null) cells were used. ( D ) Levels of miR-498 expression were measured by qRT-PCR. ( E ) MDA-MB-231 cells were transduced with the indicated expression plasmids. Colony-forming assays were performed. Ratio of colony formation is shown in the right panel as indicated. ( F ) Similar to E, except that BT-549 cells were used. All experiments were performed in triplicate. * P
Figure Legend Snippet: miR-498 promotes cell proliferation and cell cycle arrest by suppressing PTEN and increasing p-Akt levels in TNBC cells. ( A ) MDA-MB-231 cells were transduced with the indicated expression plasmids and analyzed by immunoblotting with specific antibodies. ( B ) Levels of miR-498 expression were measured by qRT-PCR. ( C ) Similar to A, except that BT-549 (PTEN null) cells were used. ( D ) Levels of miR-498 expression were measured by qRT-PCR. ( E ) MDA-MB-231 cells were transduced with the indicated expression plasmids. Colony-forming assays were performed. Ratio of colony formation is shown in the right panel as indicated. ( F ) Similar to E, except that BT-549 cells were used. All experiments were performed in triplicate. * P

Techniques Used: Multiple Displacement Amplification, Transduction, Expressing, Quantitative RT-PCR

27) Product Images from "Bmal1 suppresses cancer cell invasion by blocking the phosphoinositide 3-kinase-Akt-MMP-2 signaling pathway"

Article Title: Bmal1 suppresses cancer cell invasion by blocking the phosphoinositide 3-kinase-Akt-MMP-2 signaling pathway

Journal: Oncology Reports

doi: 10.3892/or.2013.2381

Bmal1 suppresses the PI3K-Akt-MMP-2 pathway. (A) Cell lysates from control or Bmal1-knockdown A549 cells were analyzed for PI3K activity by competitive ELISA. (B) The levels of Bmal1, the p85 subunits of PI3K, PTEN, Akt, phospho-Akt, and β-actin in the lysates were analyzed by western blotting. Alternatively, conditioned media were prepared using Bmal1-knockdown cells, and MMP-2 levels were analyzed by western blotting. Protein loading was verified by Ponceau S staining. (C) Control and Bmal1-knockdown cells were incubated in the presence or absence of LY294002 (5 μM), Akt inhibitor (5 μM), or OA-Hy (10 μM) for 24 h, and cellular invasiveness was compared. * P
Figure Legend Snippet: Bmal1 suppresses the PI3K-Akt-MMP-2 pathway. (A) Cell lysates from control or Bmal1-knockdown A549 cells were analyzed for PI3K activity by competitive ELISA. (B) The levels of Bmal1, the p85 subunits of PI3K, PTEN, Akt, phospho-Akt, and β-actin in the lysates were analyzed by western blotting. Alternatively, conditioned media were prepared using Bmal1-knockdown cells, and MMP-2 levels were analyzed by western blotting. Protein loading was verified by Ponceau S staining. (C) Control and Bmal1-knockdown cells were incubated in the presence or absence of LY294002 (5 μM), Akt inhibitor (5 μM), or OA-Hy (10 μM) for 24 h, and cellular invasiveness was compared. * P

Techniques Used: Activity Assay, Competitive ELISA, Western Blot, Staining, Incubation

28) Product Images from "Bacterial Cyclodipeptides Target Signal Pathways Involved in Malignant Melanoma"

Article Title: Bacterial Cyclodipeptides Target Signal Pathways Involved in Malignant Melanoma

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2020.01111

Proteomic analysis of CDPs effect on xenografted tumor melanoma B16-F0 cells in mice. Tumors or healthy skin were homogenized to obtain the tissue lysates used for Western blot assays as described in Materials and Methods. (A) Representative images correspond to antibody microarrays (PathScan Cancer Phenotype Antibody Array). The arrays contained the following antibodies: (1) positive control, (2) negative control, (3) CD31, (4) EpCAM, (5) vimentin, (6) CD44, (7) CD45, (8) PCNA, (9) Ki-67, (10) p27Kip1, (11) E-cadherin, (12) N-cadherin, (13) VE-cadherin, (14) MUC1, (15) Rb Ser807/811, (16) HIF-1α, (17) survivin, (18) P53, (19) HER2/ErbB2, (20) Met, (21) EGF. (B) Representative images correspond to antibody micro-arrays (PathScan Intracellular Signaling Array). The arrays contained the following antibodies: (1) positive control, (2) negative control, (3) ERK1/2-Thr202/Tyr204, (4) Stat1-Tyr701, (5) Stat3-Tyr705, (6) Akt-Thr308, (7) Akt-Ser473, (8) AMPKα-Thr172, (9) S6 Ribosomal Protein-Ser235/236, (10) mTOR-Ser2448, (11) HSP27-Ser78, (12) Bad-Ser112, (13) p70S6 Kinase-Thr389, (14) PRAS40-Thr246, (15) p53-Ser15, (16) p38-Thr180/Tyr182, (17) SAPK/JNK-Thr183/Tyr185, (18) PARP-Asp214, (19) caspase-3–Asp175, (20) GSK-3β-Ser9. Proteins that showed differences in expression/phosphorylation levels compared to the control protein extract are indicated. (C,D) Determination of signal intensity of spots from microarrays ( A,B , respectively), was conducted by densitometry using ImageJ software (NIH). Data represent the means ± SE of at two independent assays with spot duplication for each antibody, using protein extracts obtained from at least three tumors of each mouse group. Bars represent means ± SE of four densitometry determinations. One-way ANOVA was carried out, with Bonferroni post-hoc test; statistical significance ( P
Figure Legend Snippet: Proteomic analysis of CDPs effect on xenografted tumor melanoma B16-F0 cells in mice. Tumors or healthy skin were homogenized to obtain the tissue lysates used for Western blot assays as described in Materials and Methods. (A) Representative images correspond to antibody microarrays (PathScan Cancer Phenotype Antibody Array). The arrays contained the following antibodies: (1) positive control, (2) negative control, (3) CD31, (4) EpCAM, (5) vimentin, (6) CD44, (7) CD45, (8) PCNA, (9) Ki-67, (10) p27Kip1, (11) E-cadherin, (12) N-cadherin, (13) VE-cadherin, (14) MUC1, (15) Rb Ser807/811, (16) HIF-1α, (17) survivin, (18) P53, (19) HER2/ErbB2, (20) Met, (21) EGF. (B) Representative images correspond to antibody micro-arrays (PathScan Intracellular Signaling Array). The arrays contained the following antibodies: (1) positive control, (2) negative control, (3) ERK1/2-Thr202/Tyr204, (4) Stat1-Tyr701, (5) Stat3-Tyr705, (6) Akt-Thr308, (7) Akt-Ser473, (8) AMPKα-Thr172, (9) S6 Ribosomal Protein-Ser235/236, (10) mTOR-Ser2448, (11) HSP27-Ser78, (12) Bad-Ser112, (13) p70S6 Kinase-Thr389, (14) PRAS40-Thr246, (15) p53-Ser15, (16) p38-Thr180/Tyr182, (17) SAPK/JNK-Thr183/Tyr185, (18) PARP-Asp214, (19) caspase-3–Asp175, (20) GSK-3β-Ser9. Proteins that showed differences in expression/phosphorylation levels compared to the control protein extract are indicated. (C,D) Determination of signal intensity of spots from microarrays ( A,B , respectively), was conducted by densitometry using ImageJ software (NIH). Data represent the means ± SE of at two independent assays with spot duplication for each antibody, using protein extracts obtained from at least three tumors of each mouse group. Bars represent means ± SE of four densitometry determinations. One-way ANOVA was carried out, with Bonferroni post-hoc test; statistical significance ( P

Techniques Used: Mouse Assay, Western Blot, Ab Array, Positive Control, Negative Control, Expressing, Software

Effects of CDPs on the levels of proteins involved in signaling pathways promoting mouse melanoma growth. Tumor tissue was disrupted, and cell-free protein extracts were used for Western blot assays as described in Materials and Methods. Images correspond to a representative Western blot assay ( n = 3) using protein extracts of at least three tumors homogenized from the mentioned mouse groups, using the indicated antibodies. (A) Antibodies recognized p-Akt-S473, Akt, p-mTOR-S2448, mTOR, pS6K-T389, S6K, XIAP, PDK1, NFκB p65, TNF-α, and β-actin. (B) Antibodies used were against CD44, Oct3/4, C-Myc, Ras, SNAIL, MMP-1, E-CAD, VIM, CK-1, and α-tubulin. On the right, graphs correspond to determination of the band intensity from the Western blot assay (left), analyzed by densitometry using the ImageJ software. Data represent the means ± SE of densitometry determinations, n = 3 for each antibody, using protein extracts obtained from tumors of each group vs. load control (β-actin or α-tubulin). One-way ANOVA with Bonferroni post-hoc test was used to compare treatments with respect to Tumor group. Significant differences ( P
Figure Legend Snippet: Effects of CDPs on the levels of proteins involved in signaling pathways promoting mouse melanoma growth. Tumor tissue was disrupted, and cell-free protein extracts were used for Western blot assays as described in Materials and Methods. Images correspond to a representative Western blot assay ( n = 3) using protein extracts of at least three tumors homogenized from the mentioned mouse groups, using the indicated antibodies. (A) Antibodies recognized p-Akt-S473, Akt, p-mTOR-S2448, mTOR, pS6K-T389, S6K, XIAP, PDK1, NFκB p65, TNF-α, and β-actin. (B) Antibodies used were against CD44, Oct3/4, C-Myc, Ras, SNAIL, MMP-1, E-CAD, VIM, CK-1, and α-tubulin. On the right, graphs correspond to determination of the band intensity from the Western blot assay (left), analyzed by densitometry using the ImageJ software. Data represent the means ± SE of densitometry determinations, n = 3 for each antibody, using protein extracts obtained from tumors of each group vs. load control (β-actin or α-tubulin). One-way ANOVA with Bonferroni post-hoc test was used to compare treatments with respect to Tumor group. Significant differences ( P

Techniques Used: Western Blot, Software

29) Product Images from "Isorhynchophylline, a Potent Plant Alkaloid, Induces Apoptotic and Anti-Metastatic Effects in Human Hepatocellular Carcinoma Cells through the Modulation of Diverse Cell Signaling Cascades"

Article Title: Isorhynchophylline, a Potent Plant Alkaloid, Induces Apoptotic and Anti-Metastatic Effects in Human Hepatocellular Carcinoma Cells through the Modulation of Diverse Cell Signaling Cascades

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18051095

Effect of Rhy on various signal transduction pathways. ( A ) HepG2 cells (1 × 10 6 cells/well) were incubated with Rhy (130 µM) for 12 h. Whole-cell extracts were prepared, and 20 µg of the whole-cell lysate was resolved by sodium dodecyl sulfate polyacrylamide gelelectrophoresis, electrotransferred to nitrocellulose membrane, sliced from the membrane based on the molecular weight, and then probed with antibodies against p-p38, p38, p-ERK, ERK, p-JNK, and JNK as described in Materials and Methods. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. The results shown here are representative of three independent experiments; ( B ) HepG2 cells (1 × 10 6 cells/well) were treated with Rhy (130 µM) for 12 h. After that, whole-cell extracts and nuclear proteins were extract were equal amounts of lysates were analyzed by Western blot analysis using antibodies against p-CREB, CREB, p-c-Jun, and c-Jun. The same blots were stripped and reprobed with β-actin and Lamin B antibody to verify equal protein loading. The results shown here are representative of three independent experiments; ( C ) HepG2 cells (1 × 10 6 cells/well) were incubated with Rhy (130 µM) for 12 h. Whole-cell extracts were prepared, and 20 µg of the whole-cell lysate was resolved by sodium dodecyl sulfate polyacrylamide gelelectrophoresis, electrotransferred to nitrocellulose membrane, sliced from the membrane based on the molecular weight, and then probed with antibodies against p-Akt, Akt, p-STAT3, and STAT3 as described in Materials and Methods. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. The results shown here are representative of three independent experiments; ( D ) HepG2 cells (1 × 10 6 cells/well) were incubated with Rhy (130 µM) for 12 h. Whole-cell extracts were prepared, and 20 µg of the whole-cell lysate was resolved by sodium dodecyl sulfate polyacrylamide gelelectrophoresis, electrotransferred to nitrocellulose membrane, sliced from the membrane based on the molecular weight, and then probed with antibodies against p-p53, and p53 as described in Materials and Methods. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading.
Figure Legend Snippet: Effect of Rhy on various signal transduction pathways. ( A ) HepG2 cells (1 × 10 6 cells/well) were incubated with Rhy (130 µM) for 12 h. Whole-cell extracts were prepared, and 20 µg of the whole-cell lysate was resolved by sodium dodecyl sulfate polyacrylamide gelelectrophoresis, electrotransferred to nitrocellulose membrane, sliced from the membrane based on the molecular weight, and then probed with antibodies against p-p38, p38, p-ERK, ERK, p-JNK, and JNK as described in Materials and Methods. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. The results shown here are representative of three independent experiments; ( B ) HepG2 cells (1 × 10 6 cells/well) were treated with Rhy (130 µM) for 12 h. After that, whole-cell extracts and nuclear proteins were extract were equal amounts of lysates were analyzed by Western blot analysis using antibodies against p-CREB, CREB, p-c-Jun, and c-Jun. The same blots were stripped and reprobed with β-actin and Lamin B antibody to verify equal protein loading. The results shown here are representative of three independent experiments; ( C ) HepG2 cells (1 × 10 6 cells/well) were incubated with Rhy (130 µM) for 12 h. Whole-cell extracts were prepared, and 20 µg of the whole-cell lysate was resolved by sodium dodecyl sulfate polyacrylamide gelelectrophoresis, electrotransferred to nitrocellulose membrane, sliced from the membrane based on the molecular weight, and then probed with antibodies against p-Akt, Akt, p-STAT3, and STAT3 as described in Materials and Methods. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. The results shown here are representative of three independent experiments; ( D ) HepG2 cells (1 × 10 6 cells/well) were incubated with Rhy (130 µM) for 12 h. Whole-cell extracts were prepared, and 20 µg of the whole-cell lysate was resolved by sodium dodecyl sulfate polyacrylamide gelelectrophoresis, electrotransferred to nitrocellulose membrane, sliced from the membrane based on the molecular weight, and then probed with antibodies against p-p53, and p53 as described in Materials and Methods. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading.

Techniques Used: Transduction, Incubation, Molecular Weight, Western Blot

30) Product Images from "Nuclear and cytoplasmic p53 suppress cell invasion by inhibiting respiratory Complex-I activity via Bcl-2 family proteins"

Article Title: Nuclear and cytoplasmic p53 suppress cell invasion by inhibiting respiratory Complex-I activity via Bcl-2 family proteins

Journal: Oncotarget

doi:

Cytoplasmic p53 suppresses cell invasion by binding to Bcl-w and liberating Bax from Bcl-w (A) Empty pcDNA3 or vectors encoding Bcl-w, p53, p53 K305N , and p53 K305N/R175H were introduced into H1299 cells. Gene expression was confirmed by Western blotting with β-actin as a loading control. Invasiveness was assessed on Matrigel-coated filters. ns, not significant. (B) Transfectants were analyzed for cellular and mitochondrial ROS using DCF and MitoSOX Red probes, respectively. Flow cytometry profiles of key experiments are shown on top. (C) Lysates were prepared and immunoprecipitated with an anti-PI3K antibody. PI3K activity was analyzed by competitive ELISA. Lysates were also analyzed for Akt and phospho-Akt by Western blotting. Conditioned media were prepared and analyzed for MMP-2 levels by Western blotting. Protein loading for conditioned media was verified by Ponceau S staining. (D) Wild-type and mutant p53 proteins were prepared by in vitro translation and incubated with GST, GST-Bcl-w, or GST-Bax proteins. Precipitation was carried out with glutathione-coupled Sepharose beads. Precipitates and input controls were analyzed by Western blotting with anti-p53 antibody. (E) Lysates were immunoprecipitated with anti-Bcl-w or anti-Bax antibodies. Precipitates and input controls were analyzed by Western blotting. (F) Bax and Bcl-w, together with p53 or p53 mutants, were translated in vitro . The products were verified by Western blotting (input) and immunoprecipitated with anti-Bax antibody. Levels of Bcl-w in precipitates were compared.
Figure Legend Snippet: Cytoplasmic p53 suppresses cell invasion by binding to Bcl-w and liberating Bax from Bcl-w (A) Empty pcDNA3 or vectors encoding Bcl-w, p53, p53 K305N , and p53 K305N/R175H were introduced into H1299 cells. Gene expression was confirmed by Western blotting with β-actin as a loading control. Invasiveness was assessed on Matrigel-coated filters. ns, not significant. (B) Transfectants were analyzed for cellular and mitochondrial ROS using DCF and MitoSOX Red probes, respectively. Flow cytometry profiles of key experiments are shown on top. (C) Lysates were prepared and immunoprecipitated with an anti-PI3K antibody. PI3K activity was analyzed by competitive ELISA. Lysates were also analyzed for Akt and phospho-Akt by Western blotting. Conditioned media were prepared and analyzed for MMP-2 levels by Western blotting. Protein loading for conditioned media was verified by Ponceau S staining. (D) Wild-type and mutant p53 proteins were prepared by in vitro translation and incubated with GST, GST-Bcl-w, or GST-Bax proteins. Precipitation was carried out with glutathione-coupled Sepharose beads. Precipitates and input controls were analyzed by Western blotting with anti-p53 antibody. (E) Lysates were immunoprecipitated with anti-Bcl-w or anti-Bax antibodies. Precipitates and input controls were analyzed by Western blotting. (F) Bax and Bcl-w, together with p53 or p53 mutants, were translated in vitro . The products were verified by Western blotting (input) and immunoprecipitated with anti-Bax antibody. Levels of Bcl-w in precipitates were compared.

Techniques Used: Binding Assay, Expressing, Western Blot, Flow Cytometry, Cytometry, Immunoprecipitation, Activity Assay, Competitive ELISA, Staining, Mutagenesis, In Vitro, Incubation

31) Product Images from "Micro RNA‐20a‐5p contributes to hepatic glycogen synthesis through targeting p63 to regulate p53 and PTEN expression"

Article Title: Micro RNA‐20a‐5p contributes to hepatic glycogen synthesis through targeting p63 to regulate p53 and PTEN expression

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12835

MiR‐20a‐5p contributes to glycogenesis in hepatocytes. The levels of miR‐20a‐5p ( A ), glycogen synthesis ( B and C ) and phosphorylation of AKT and GSK ( D and E ) were analysed in NCTC 1469 cells and Hep1‐6 cells transfected with miR‐20a‐5p inhibitor. The transfected efficiency of miR‐20a‐5p mimic was measured in NCTC 1469 cells and Hep1‐6 cells ( F ). Moreover, miR‐20a‐5p mimic was transfected in NCTC 1469 cells and Hep1‐6 cells treated with 33.3 mmol/l glucose for 48 hrs. The levels of glycogen synthesis ( G and H ) and phosphorylation of AKT and GSK ( I and J ) were measured. Data represent the mean ± S.D. N = 3 independent experiments. * P
Figure Legend Snippet: MiR‐20a‐5p contributes to glycogenesis in hepatocytes. The levels of miR‐20a‐5p ( A ), glycogen synthesis ( B and C ) and phosphorylation of AKT and GSK ( D and E ) were analysed in NCTC 1469 cells and Hep1‐6 cells transfected with miR‐20a‐5p inhibitor. The transfected efficiency of miR‐20a‐5p mimic was measured in NCTC 1469 cells and Hep1‐6 cells ( F ). Moreover, miR‐20a‐5p mimic was transfected in NCTC 1469 cells and Hep1‐6 cells treated with 33.3 mmol/l glucose for 48 hrs. The levels of glycogen synthesis ( G and H ) and phosphorylation of AKT and GSK ( I and J ) were measured. Data represent the mean ± S.D. N = 3 independent experiments. * P

Techniques Used: Transfection

P63 regulates expression of PTEN by directly binding to p53. A direct interaction between p63 and p53 was observed by co‐immunoprecipitation ( A ). The levels of p63, p53 and PTEN protein were measured in the liver of db/db mice ( B ), NCTC 1469 cells ( C ) and Hep1‐6 cells ( D ) treated with high glucose as well as NCTC 1469 cells ( E ) and Hep1‐6 cells ( F ) transfected with si RNA targeting p63. Overexpression of miR‐20a‐5p led to decreased protein levels of p53 and PTEN ( G and H ), whereas inhibition of miR‐20a‐5p increased the expression of p53 and PTEN ( I and J ). Transfection of miR‐20a‐5p mimic significantly decreased the fluorescence intensity of p53 and PTEN , as shown by immunofluorescence assay ( K ). Moreover, overexpression of miR‐20a‐5p in NCTC 1469 cells and Hep1‐6 cells could reverse high glucose‐induced increased expression of p63, p53 and PTEN ( L and M ). Moreover, p53 knockdown could reverse miR‐20a‐5p inhibition‐induced reduced expression of genes related to hepatic glycogen synthesis such as UPG 2, G6 PC and GBE 1 ( N ) and hepatic glycogen synthesis ( O ), and activation of AKT and GSK ( P ) in the NCTC 1469 cells treated by miR‐20a‐5p inhibition. Scale bar represents 20 μm. Data represent the mean ± S.D. N = 5 mice or N = 3 independent experiments. * P
Figure Legend Snippet: P63 regulates expression of PTEN by directly binding to p53. A direct interaction between p63 and p53 was observed by co‐immunoprecipitation ( A ). The levels of p63, p53 and PTEN protein were measured in the liver of db/db mice ( B ), NCTC 1469 cells ( C ) and Hep1‐6 cells ( D ) treated with high glucose as well as NCTC 1469 cells ( E ) and Hep1‐6 cells ( F ) transfected with si RNA targeting p63. Overexpression of miR‐20a‐5p led to decreased protein levels of p53 and PTEN ( G and H ), whereas inhibition of miR‐20a‐5p increased the expression of p53 and PTEN ( I and J ). Transfection of miR‐20a‐5p mimic significantly decreased the fluorescence intensity of p53 and PTEN , as shown by immunofluorescence assay ( K ). Moreover, overexpression of miR‐20a‐5p in NCTC 1469 cells and Hep1‐6 cells could reverse high glucose‐induced increased expression of p63, p53 and PTEN ( L and M ). Moreover, p53 knockdown could reverse miR‐20a‐5p inhibition‐induced reduced expression of genes related to hepatic glycogen synthesis such as UPG 2, G6 PC and GBE 1 ( N ) and hepatic glycogen synthesis ( O ), and activation of AKT and GSK ( P ) in the NCTC 1469 cells treated by miR‐20a‐5p inhibition. Scale bar represents 20 μm. Data represent the mean ± S.D. N = 5 mice or N = 3 independent experiments. * P

Techniques Used: Expressing, Binding Assay, Immunoprecipitation, Mouse Assay, Transfection, Over Expression, Inhibition, Fluorescence, Immunofluorescence, Activation Assay

Knockdown of p63 improves glycogen synthesis. Two specific si RNA targeting p63 was selected ( A ). Genes related to hepatic glycogen synthesis, glycogen content ( B and C ) and phosphorylation levels of AKT and GSK ( D and E ) were measured in the NCTC 1469 cells and the Hep1‐6 cells transfected with si RNA targeting p63. Moreover, p63 knockdown could reverse miR‐20a‐5p inhibition‐induced reduced glycogenesis and activation of AKT and GSK ( F and G ). Data represent the mean ± S.D. N = 3 independent experiments. * P
Figure Legend Snippet: Knockdown of p63 improves glycogen synthesis. Two specific si RNA targeting p63 was selected ( A ). Genes related to hepatic glycogen synthesis, glycogen content ( B and C ) and phosphorylation levels of AKT and GSK ( D and E ) were measured in the NCTC 1469 cells and the Hep1‐6 cells transfected with si RNA targeting p63. Moreover, p63 knockdown could reverse miR‐20a‐5p inhibition‐induced reduced glycogenesis and activation of AKT and GSK ( F and G ). Data represent the mean ± S.D. N = 3 independent experiments. * P

Techniques Used: Transfection, Inhibition, Activation Assay

Down‐regulation of miR‐20a‐5p is accompanied by reduced glycogen synthesis. The levels of miR‐20a‐5p and other five members of miR‐17 family including miR‐20b‐5p, miR‐106a‐5p, miR‐106b‐5p, miR‐17‐5p and miR‐93‐5p were measured in the liver of db/db mice ( A ). The level of miR‐20a‐5p was detected in patients with NAFLD ( B ). Murine NCTC 1469 and Hep1‐6 hepatocytes were stimulated with 33.3 mmol/l glucose for 48 hrs, 0.25 mmol/l palmitate for 24 hrs, 10 nmol/l IL ‐6 or 10 nmol/l TNF ‐α for 24 hrs respectively. The level of miR‐20a‐5p was determined in both cells ( C ). The changes of miR‐17 family members were detected in NCTC 1469 and Hep1‐6 cells stimulated with 33.3 mmol/l glucose for 48 hrs ( D ). Genes related to hepatic glycogen synthesis, glycogen level ( E and F ) and activation of AKT and GSK ( G and H ) were analysed in NCTC 1469 and Hep1‐6cells treated with 33.3 mmol/l glucose for 48 hrs. Data represent the mean ± S.D. N = 5 mice or N = 3 independent experiments. * P
Figure Legend Snippet: Down‐regulation of miR‐20a‐5p is accompanied by reduced glycogen synthesis. The levels of miR‐20a‐5p and other five members of miR‐17 family including miR‐20b‐5p, miR‐106a‐5p, miR‐106b‐5p, miR‐17‐5p and miR‐93‐5p were measured in the liver of db/db mice ( A ). The level of miR‐20a‐5p was detected in patients with NAFLD ( B ). Murine NCTC 1469 and Hep1‐6 hepatocytes were stimulated with 33.3 mmol/l glucose for 48 hrs, 0.25 mmol/l palmitate for 24 hrs, 10 nmol/l IL ‐6 or 10 nmol/l TNF ‐α for 24 hrs respectively. The level of miR‐20a‐5p was determined in both cells ( C ). The changes of miR‐17 family members were detected in NCTC 1469 and Hep1‐6 cells stimulated with 33.3 mmol/l glucose for 48 hrs ( D ). Genes related to hepatic glycogen synthesis, glycogen level ( E and F ) and activation of AKT and GSK ( G and H ) were analysed in NCTC 1469 and Hep1‐6cells treated with 33.3 mmol/l glucose for 48 hrs. Data represent the mean ± S.D. N = 5 mice or N = 3 independent experiments. * P

Techniques Used: Mouse Assay, Activation Assay

32) Product Images from "Glycolipid Metabolism Disorder in the Liver of Obese Mice Is Improved by TUDCA via the Restoration of Defective Hepatic Autophagy"

Article Title: Glycolipid Metabolism Disorder in the Liver of Obese Mice Is Improved by TUDCA via the Restoration of Defective Hepatic Autophagy

Journal: International Journal of Endocrinology

doi: 10.1155/2015/687938

Effects of TUDCA on hepatic autophagic response, ER stress, and insulin signaling in the liver of obese mice. All analyses were performed in mice fed a normal diet (ND) or a high-fat diet (HFD) for 8 weeks and then injected daily with 500 mg/kg of TUDCA or with vehicle (Veh) for 8 weeks. (a) Protein expression of Atg7, p62, and LC3 in liver. (b) The relative protein quantity of Atg7, p62, and LC3 in liver. (c) Quantification of autophagolysosome-like vacuoles per field in the EM images of liver (magnification 40000x). (d) Quantification of autophagolysosome-like vacuoles per field in the EM images of liver. (e) Phosphorylation of PERK and eIF2 α in liver. (f) The relative protein quantity of p-PERK and p-eIF2 α in liver. (g) Phosphorylation of IR and Akt in liver. (h) The relative protein quantity of p-IR and p-Akt in liver. The relative quantity of proteins was analyzed using Quantity One software. A representative blot is shown and the data was expressed as mean ± SEM in each bar graph. ∗ P
Figure Legend Snippet: Effects of TUDCA on hepatic autophagic response, ER stress, and insulin signaling in the liver of obese mice. All analyses were performed in mice fed a normal diet (ND) or a high-fat diet (HFD) for 8 weeks and then injected daily with 500 mg/kg of TUDCA or with vehicle (Veh) for 8 weeks. (a) Protein expression of Atg7, p62, and LC3 in liver. (b) The relative protein quantity of Atg7, p62, and LC3 in liver. (c) Quantification of autophagolysosome-like vacuoles per field in the EM images of liver (magnification 40000x). (d) Quantification of autophagolysosome-like vacuoles per field in the EM images of liver. (e) Phosphorylation of PERK and eIF2 α in liver. (f) The relative protein quantity of p-PERK and p-eIF2 α in liver. (g) Phosphorylation of IR and Akt in liver. (h) The relative protein quantity of p-IR and p-Akt in liver. The relative quantity of proteins was analyzed using Quantity One software. A representative blot is shown and the data was expressed as mean ± SEM in each bar graph. ∗ P

Techniques Used: Mouse Assay, Injection, Expressing, Software

Improvement of ER stress and insulin signaling by the restoration of Atg7 in liver of obese mice. All analyses were performed in mice fed a high-fat diet (HFD) for 8 weeks and then adenovirus carrying Atg7 or GFP was delivered into obese mice via orbital venous plexus at a titer of 3 × 10 11 vp/mice. (a) Protein expression of Atg7, p62, and LC3 in liver. (b) The relative protein quantity of Atg7, p62, and LC3 in liver. (c) Quantification of autophagolysosome-like vacuoles per field in the EM images of liver (magnification 40000x). (d) Quantification of autophagolysosome-like vacuoles per field in the EM images of liver. (e) Phosphorylation of PERK and eIF2 α in liver. (f) The relative protein quantity of p-PERK and p-eIF2 α in liver. (g) Phosphorylation of IR and Akt in liver. (h) The relative protein quantity of p-IR and p-Akt in liver. (i) GTT. (j) Area under the curve by GTT. (k) ITT. (l) Area under the curve by ITT. The relative quantity of proteins was analyzed using Quantity One software. A representative blot is shown and the data was expressed as mean ± SEM in each bar graph. ∗ P
Figure Legend Snippet: Improvement of ER stress and insulin signaling by the restoration of Atg7 in liver of obese mice. All analyses were performed in mice fed a high-fat diet (HFD) for 8 weeks and then adenovirus carrying Atg7 or GFP was delivered into obese mice via orbital venous plexus at a titer of 3 × 10 11 vp/mice. (a) Protein expression of Atg7, p62, and LC3 in liver. (b) The relative protein quantity of Atg7, p62, and LC3 in liver. (c) Quantification of autophagolysosome-like vacuoles per field in the EM images of liver (magnification 40000x). (d) Quantification of autophagolysosome-like vacuoles per field in the EM images of liver. (e) Phosphorylation of PERK and eIF2 α in liver. (f) The relative protein quantity of p-PERK and p-eIF2 α in liver. (g) Phosphorylation of IR and Akt in liver. (h) The relative protein quantity of p-IR and p-Akt in liver. (i) GTT. (j) Area under the curve by GTT. (k) ITT. (l) Area under the curve by ITT. The relative quantity of proteins was analyzed using Quantity One software. A representative blot is shown and the data was expressed as mean ± SEM in each bar graph. ∗ P

Techniques Used: Mouse Assay, Expressing, Software

33) Product Images from "sMEK1 inhibits endothelial cell proliferation by attenuating VEGFR-2-dependent-Akt/eNOS/HIF-1α signaling pathways"

Article Title: sMEK1 inhibits endothelial cell proliferation by attenuating VEGFR-2-dependent-Akt/eNOS/HIF-1α signaling pathways

Journal: Oncotarget

doi:

sMEK1 suppressed the phosphorylated proteins of the VEGFR-2/PI3K/eNOS signaling cascade Cell lysates were prepared from transfected SKOV-3 cancer cells or HUVECs (data not shown) and subjected to immunoblotting using primary antibodies specific to the phosphorylated or unphosphorylated forms of negative and positive regulators of Akt, including PI3K and eNOS. All experiments were repeated at least three times with similar results.
Figure Legend Snippet: sMEK1 suppressed the phosphorylated proteins of the VEGFR-2/PI3K/eNOS signaling cascade Cell lysates were prepared from transfected SKOV-3 cancer cells or HUVECs (data not shown) and subjected to immunoblotting using primary antibodies specific to the phosphorylated or unphosphorylated forms of negative and positive regulators of Akt, including PI3K and eNOS. All experiments were repeated at least three times with similar results.

Techniques Used: Transfection

34) Product Images from "miR-144 suppresses the growth and metastasis of laryngeal squamous cell carcinoma by targeting IRS1"

Article Title: miR-144 suppresses the growth and metastasis of laryngeal squamous cell carcinoma by targeting IRS1

Journal: American Journal of Translational Research

doi:

IRS1 is a direct target of miR-144a. A. The predicted binding sites for miR-144 in the 3’UTR of IRS1 and the mutations in the binding sites are shown. B. The luciferase activity of the wild type IRS1 3-UTR (Wt) and mutant IRS1 3’UTR (Mut) co-transfected with miR-144 or miR-NC was determined. C. IRS1 expression on mRNA level in Hep2 cells transfected with miR-144 mimic or miR-NC were determined by qRT-PCR. D. IRS1, AKT and p-AKT protein expression were determined in Hep2 cells transfected with miR-144 mimic or miR-NC by western blot. qRT-PCR and western blot data were normalized to β-actin. *P
Figure Legend Snippet: IRS1 is a direct target of miR-144a. A. The predicted binding sites for miR-144 in the 3’UTR of IRS1 and the mutations in the binding sites are shown. B. The luciferase activity of the wild type IRS1 3-UTR (Wt) and mutant IRS1 3’UTR (Mut) co-transfected with miR-144 or miR-NC was determined. C. IRS1 expression on mRNA level in Hep2 cells transfected with miR-144 mimic or miR-NC were determined by qRT-PCR. D. IRS1, AKT and p-AKT protein expression were determined in Hep2 cells transfected with miR-144 mimic or miR-NC by western blot. qRT-PCR and western blot data were normalized to β-actin. *P

Techniques Used: Binding Assay, Luciferase, Activity Assay, Mutagenesis, Transfection, Expressing, Quantitative RT-PCR, Western Blot

35) Product Images from "FoxO3a Nuclear Localization and Its Association with β-Catenin and Smads in IFN-α-Treated Hepatocellular Carcinoma Cell Lines"

Article Title: FoxO3a Nuclear Localization and Its Association with β-Catenin and Smads in IFN-α-Treated Hepatocellular Carcinoma Cell Lines

Journal: Journal of Interferon & Cytokine Research

doi: 10.1089/jir.2013.0124

Regulation of Akt, IκB kinase β (IKKβ), extracellular-signal-regulated kinase (Erk), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated kinase (p38 MAPK) by IFN-α2b in HCC cell lines. C3A and Huh7 cells were incubated
Figure Legend Snippet: Regulation of Akt, IκB kinase β (IKKβ), extracellular-signal-regulated kinase (Erk), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated kinase (p38 MAPK) by IFN-α2b in HCC cell lines. C3A and Huh7 cells were incubated

Techniques Used: Incubation

36) Product Images from "Chitosan oligosaccharides suppress LPS-induced IL-8 expression in human umbilical vein endothelial cells through blockade of p38 and Akt protein kinases"

Article Title: Chitosan oligosaccharides suppress LPS-induced IL-8 expression in human umbilical vein endothelial cells through blockade of p38 and Akt protein kinases

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2011.10

Inhibitory effect of COS on LPS-induced over-expression of phosphorylated p38 MAPK (A) and Akt (B) in HUVECs. Cells were pretreated with COS (50, 100, and 200 μg/mL) for 24 h and then exposed to LPS (100 ng/mL) for 10 min for p38 detection and
Figure Legend Snippet: Inhibitory effect of COS on LPS-induced over-expression of phosphorylated p38 MAPK (A) and Akt (B) in HUVECs. Cells were pretreated with COS (50, 100, and 200 μg/mL) for 24 h and then exposed to LPS (100 ng/mL) for 10 min for p38 detection and

Techniques Used: Over Expression

37) Product Images from "Downregulation of MiR-203a Disinhibits Bmi1 and Promotes Growth and Proliferation of Keratinocytes in Cholesteatoma"

Article Title: Downregulation of MiR-203a Disinhibits Bmi1 and Promotes Growth and Proliferation of Keratinocytes in Cholesteatoma

Journal: International Journal of Medical Sciences

doi: 10.7150/ijms.22410

Overexpression of p-Akt is positively correlated with Bmi1 in cholesteatoma. (A) Western blot analysis of p-Akt, total Akt, and Bmi1 expression in 20 paired cholesteatoma and retroauricular skin specimens (C, cholesteatoma; S, retroauricular skin). (B) Statistical analysis of p-Akt expression from 20 paired cholesteatoma and retroauricular skin specimens. * P
Figure Legend Snippet: Overexpression of p-Akt is positively correlated with Bmi1 in cholesteatoma. (A) Western blot analysis of p-Akt, total Akt, and Bmi1 expression in 20 paired cholesteatoma and retroauricular skin specimens (C, cholesteatoma; S, retroauricular skin). (B) Statistical analysis of p-Akt expression from 20 paired cholesteatoma and retroauricular skin specimens. * P

Techniques Used: Over Expression, Western Blot, Expressing

38) Product Images from "Relationship between polycomb‐group protein BMI‐1 and phosphatases regulating AKT phosphorylation level in endometrial cancer, et al. Relationship between polycomb‐group protein BMI‐1 and phosphatases regulating AKT phosphorylation level in endometrial cancer"

Article Title: Relationship between polycomb‐group protein BMI‐1 and phosphatases regulating AKT phosphorylation level in endometrial cancer, et al. Relationship between polycomb‐group protein BMI‐1 and phosphatases regulating AKT phosphorylation level in endometrial cancer

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14782

Representative results of Western blot analysis of BMI‐1, PTEN, AKT and phospho‐AKT levels in normal and cancer endometrial tissues. The stage, grade and lymph node metastasis status of cancers are indicated. R‐reference sample, N‐normal endometrial tissue, EC‐endometrial cancer, NS‐nodal status
Figure Legend Snippet: Representative results of Western blot analysis of BMI‐1, PTEN, AKT and phospho‐AKT levels in normal and cancer endometrial tissues. The stage, grade and lymph node metastasis status of cancers are indicated. R‐reference sample, N‐normal endometrial tissue, EC‐endometrial cancer, NS‐nodal status

Techniques Used: Western Blot

Effect of BMI‐1 down‐regulation on AKT phosphorylation level and expression of phosphatases in HEC1A cells. A, Representative immunoblots showing BMI‐1, PTEN, AKT proteins and phosphorylated AKT in HEC1A cells treated for 48 h with 30 nmol/L BMI‐1 siRNA or scrambled siRNA (control). B, Bar graph shows the densitometric analysis of BMI‐1, PTEN, AKT and phosphorylated AKT (pAKT) levels in cells treated with BMI‐1 siRNA and scrambled siRNA and represents the mean ± SD of three independent experiments. C, Relative changes in BMI1, PP2A, PHLPP1, PHLPP2, INPP4B, INPP5D and PTEN mRNAs expression levels in siRNA treated cells compared to untreated cells; bar graph represents the mean ± SD. * P
Figure Legend Snippet: Effect of BMI‐1 down‐regulation on AKT phosphorylation level and expression of phosphatases in HEC1A cells. A, Representative immunoblots showing BMI‐1, PTEN, AKT proteins and phosphorylated AKT in HEC1A cells treated for 48 h with 30 nmol/L BMI‐1 siRNA or scrambled siRNA (control). B, Bar graph shows the densitometric analysis of BMI‐1, PTEN, AKT and phosphorylated AKT (pAKT) levels in cells treated with BMI‐1 siRNA and scrambled siRNA and represents the mean ± SD of three independent experiments. C, Relative changes in BMI1, PP2A, PHLPP1, PHLPP2, INPP4B, INPP5D and PTEN mRNAs expression levels in siRNA treated cells compared to untreated cells; bar graph represents the mean ± SD. * P

Techniques Used: Expressing, Western Blot

39) Product Images from "Involvement of the Na+/Ca2+ exchanger isoform 1 (NCX1) in Neuronal Growth Factor (NGF)-induced Neuronal Differentiation through Ca2+-dependent Akt Phosphorylation *"

Article Title: Involvement of the Na+/Ca2+ exchanger isoform 1 (NCX1) in Neuronal Growth Factor (NGF)-induced Neuronal Differentiation through Ca2+-dependent Akt Phosphorylation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.555516

Effect of ERK1/2 modulation on [Ca 2+ ] i homeostasis, Akt phosphorylation, and GAP-43 protein expression in NGF-induced PC12 differentiation. A and B , representative Western blot and relative quantification of ERK1/2 in PC12 cells exposed to NGF for 5 min
Figure Legend Snippet: Effect of ERK1/2 modulation on [Ca 2+ ] i homeostasis, Akt phosphorylation, and GAP-43 protein expression in NGF-induced PC12 differentiation. A and B , representative Western blot and relative quantification of ERK1/2 in PC12 cells exposed to NGF for 5 min

Techniques Used: Expressing, Western Blot

40) Product Images from "Positive and Negative Regulation of Mast Cell Activation by Lyn via the Fc?RI 1"

Article Title: Positive and Negative Regulation of Mast Cell Activation by Lyn via the Fc?RI 1

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi:

Phosphorylation of ERK, p38, and Akt in wt and lyn − / − mast cells upon stimulation with low and high concentrations of Ag. Wt and lyn − / − BMMC were sensitized with IgE and stimulated with 1 or 100 ng/ml DNP 21 -BSA for the
Figure Legend Snippet: Phosphorylation of ERK, p38, and Akt in wt and lyn − / − mast cells upon stimulation with low and high concentrations of Ag. Wt and lyn − / − BMMC were sensitized with IgE and stimulated with 1 or 100 ng/ml DNP 21 -BSA for the

Techniques Used:

Related Articles

Western Blot:

Article Title: Function of the Nucleotide Exchange Activity of Vav1 in T cell Development and Activation
Article Snippet: .. The following antibodies were used for detection of proteins by Western blotting: anti-phosphotyrosine (4G10, Upstate Biotechnology), anti-phospho-ERK (E-4), anti-Vav1 (C-14), anti-Akt (B-1) from Santa Cruz Biotechnology, anti-phosphoserine473-Akt, and anti-phosphotyrosine171-human LAT (equivalent to pY175 murine LAT) from Cell Signaling Technology, anti-ERK2 (gift from C. Marshall), anti-LAT (gift from M. Turner), and anti-PKD1 and anti-phosphoserine916-PKD1 (both gifts from D. Cantrell). .. Antibody binding was revealed with IRDye800-conjugated goat antibody against mouse IgG (Rockland) for mouse antibodies, or Alexa Fluor 680–conjugated goat antibody against rabbit IgG (Invitrogen) for rabbit polyclonal antibodies.

Incubation:

Article Title: Activation of the MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis protects MG-63 osteosarcoma cells against 15d-PGJ2-mediated cell death
Article Snippet: .. For normalization, membranes were stripped with stripping buffer (58.4 g/L NaCl, 7.5 g/L glycine, pH 2.15, Sigma–Aldrich) and incubated with anti-p38 (1:2000 Sigma–Aldrich-M0800), anti-Akt (1:500 Santa Cruz-sc1618R), anti-Lamin (1:1000 Santa Cruz-sc20681), or anti-γ-actin antisera (1:1000 Santa Cruz-sc47778) as primary antibodies [ , ]. .. Annexin V/propidium iodide (PI) staining MG-63 cells (70% confluent) were treated with 20 μM 15d-PGJ2 up to 48 h. Cells were harvested and stained using the FITC Annexin V Apoptosis Detection Kit 1 (BD Biosciences, NJ, USA) according to the manufacturer’s suggestion.

Article Title: Thymoquinone reduces spinal cord injury by inhibiting inflammatory response, oxidative stress and apoptosis via PPAR-γ and PI3K/Akt pathways
Article Snippet: .. The membrane was blocked with 5% (w/v) non-fat milk in Tris-buffered saline containing 0.05% Tween-20 at 37°C for 1 h and incubated with anti-cyclooxygenase 2 (COX-2; cat. no. sc-7951, dilution 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-PPAR-γ (cat. no. sc-9000, dilution 1:1,000; Santa Cruz Biotechnology, Inc.), anti-PI3K (cat. no. sc-7175, dilution 1:1,000; Santa Cruz Biotechnology, Inc.), anti-Akt (cat. no. sc-8312, dilution 1:500; Santa Cruz Biotechnology, Inc.), anti-p-Akt (cat. no. sc-7985-R, dilution 1:1,000; Santa Cruz Biotechnology, Inc.) and anti-GAPDH (cat. no. sc-25778, dilution 1:2,000; Santa Cruz Biotechnology, Inc.) at 4°C overnight. .. The membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (dilution 1:5,000, cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) for 1 h at 37°C and visualized with an enhanced chemiluminescence system using sodium Image_Lab_3.0 (Bio-Rad Laboratories, Inc.).

Stripping Membranes:

Article Title: RasGRF1 regulates proliferation and metastatic behavior of human alveolar rhabdomyosarcomas
Article Snippet: .. Equal loading in the lanes was evaluated by stripping the blots and reprobing with appropriate mAbs: p42/44 anti-MAPK clone no. 9102 and anti-AKT clone no. 9272 (Santa Cruz Biotechnology). .. The membranes were developed with an enhanced chemiluminescence (ECL) reagent (Amersham Life Sciences, Little Chalfont, UK), dried, and exposed to film (HyperFilm, Amersham Life Sciences).

Article Title: Activation of the MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis protects MG-63 osteosarcoma cells against 15d-PGJ2-mediated cell death
Article Snippet: .. For normalization, membranes were stripped with stripping buffer (58.4 g/L NaCl, 7.5 g/L glycine, pH 2.15, Sigma–Aldrich) and incubated with anti-p38 (1:2000 Sigma–Aldrich-M0800), anti-Akt (1:500 Santa Cruz-sc1618R), anti-Lamin (1:1000 Santa Cruz-sc20681), or anti-γ-actin antisera (1:1000 Santa Cruz-sc47778) as primary antibodies [ , ]. .. Annexin V/propidium iodide (PI) staining MG-63 cells (70% confluent) were treated with 20 μM 15d-PGJ2 up to 48 h. Cells were harvested and stained using the FITC Annexin V Apoptosis Detection Kit 1 (BD Biosciences, NJ, USA) according to the manufacturer’s suggestion.

SDS Page:

Article Title: S100A8 is a Novel Therapeutic Target for Anaplastic Thyroid Carcinoma
Article Snippet: .. Protein sample was resolved by the SDS/PAGE followed by immunoblotting with anti-S100A8 (1:300, Sigma), anti-S100A9 (1:1000, Abcam), anti-p-ERK1/2 (T202/Y204) (1:2000, Cell Signaling), anti-ERK1/2 (1:4000, Cell Signaling), anti-p-SAPK/JNK (T183/Y185) (1:500, Cell Signaling), anti-SAPK/JNK (1:500, Cell Signaling), anti-p38 (1:4000; Santa Cruz), anti-p-p38 (T180/Y182) (1:1000; Santa Cruz), anti-p-AKT (Ser473) (1:500, Santa Cruz), anti-AKT (1:500, Santa Cruz), β-actin (1:2000; Sigma), and GAPDH (1:4000, Cell Signaling) antibodies. ..

Immunodetection:

Article Title: Centrosomal Protein of 55 Regulates Glucose Metabolism, Proliferation and Apoptosis of Glioma Cells via the Akt/mTOR Signaling Pathway
Article Snippet: .. Primary antibodies for immunodetection were anti-CEP55 (Santa Cruz), anti-GLUT1 (Santa Cruz), anti-p-AktS473 (Santa Cruz), anti-p-AktT308 (Santa Cruz), anti-Akt (Santa Cruz), anti-p-mTOR (Santa Cruz), anti-mTOR (Santa Cruz), anti-BAD (Santa Cruz), anti-caspase-9 (Santa Cruz), anti-GSK3-β (Santa Cruz), anti-p27 (Abcam) and anti-GAPDH (Santa Cruz). .. Subsequent to being incubated with Horseradish peroxidase (HRP) conjugated anti-rabbit or anti-mouse secondary antibodies (1: 10000, Santa Cruz) for 1 h, the immune complexes were detected using the enhanced chemiluminescence method.

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  • 92
    Santa Cruz Biotechnology anti akt
    RasGRF1 is involved in chemokine and growth factor receptor signaling. (A), Effect of RasGRF1 down-regulation on activation of intracellular signaling in ARMS cells. Phosphorylation of <t>p42/44</t> MAPK and <t>AKT</t> in RH30-derived cell lines was stimulated for 5 min by SDF-1 (300 ng/ml), I-TAC (100 ng/ml), HGF (100 ng/ml), IGF-II (100 ng/ml), and insulin (10 ng/ml). The experiment was repeated three times with similar results. A representative result is shown. (B), RasGRF1 phosphorylation after stimulation with chemokines and growth factors. RasGRF1 protein phophorylated at Ser929 was detected by western blot analysis after stimulation for 5 min by SDF-1 (300 ng/ml), I-TAC (100 ng/ml), HGF (100 ng/ml), IGF-II (100 ng/ml), and insulin (10 ng/ml). (C), Effect of RasGRF1 down-regulation on Ras activation. A Ras pull-down assay was performed on two RH30-derived cell lines (RH30scr and RH30 RasGRF1-kd). The cells were stimulated for 5 min with SDF-1 (300 ng/ml) or IGF-II (100 ng/ml). Ras-GTP was precipitated by Raf-1 RBD agarose conjugate and detected by Ras antibody clone RAS10 (Millipore). The same antibody was used to detect total Ras protein (p21 H-, K- and N-Ras). Western blots were analyzed by densitometry (right side). The experiment was repeated three times with similar results. A representative result is shown. (D), Effect of RasGRF1 down-regulation on paxillin expression and actin cytoskeleton. Staining of paxillin and actin was performed on RH30scr, RH30 RasGRF1-kd, RH18scr and RH18 RasGRF-kd cell lines. The experiment was repeated three times and representative results are shown.
    Anti Akt, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 342 article reviews
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    94
    Santa Cruz Biotechnology anti phospho akt
    Mdm20 regulates the phosphorylation status of <t>Akt.</t> A. Western blots for Akt with a focus on phosphorylated Akt on Ser473 and Thr308. HEK293 cells were transfected either with mock, Flag-Mdm20 and mCherry-Nat5 or control, Mdm20 and Nat5 siRNA. At 48 hrs or 72hrs post-transfection, respectively, the cells were harvested, and cell lysates were prepared and processed for western blot analysis. The antibodies used are indicated. For details, see Materials and Methods. B. Western blots were used to determine the phosphorylation status of GSK3β, PTEN, PDK-1, mTOR, and <t>PP1.</t> The antibodies used were as indicated. Note that the phosphorylation of mTOR (Ser-2481) is consistently reduced in Mdm20-KD cells. C. Akt inhibition increases the levels of LC3II. Shown is western blots of HEK293 cellular extracts treated or untreated with siRNAs for Mdm20 or Nat5 in the absence (DMSO control) or presence of Akt inhibitor (Akt-I-VIII). Levels of Akt, and phospho-Akt (pAkt-Ser473), and LC3 levels are shown with the loading control of actin blot.
    Anti Phospho Akt, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology anti akt pt308
    BTG3 suppresses growth of PC3 prostate cancer cells in 3D culture. ( a ) Characterization of PC3 Tet-On cells that express myc-tagged BTG3 upon the addition of doxycycline (+). TR, control cells that express only the tetracycline regulator; ovBTG3, Tet-On BTG3-overexpressing cells (clone no. 6). <t>AKT</t> T308 phosphorylation <t>(pT308)</t> was downregulated in ovBTG3 cells. ( b ) Immunofluorescence microscopy of spheroids grown in 3D culture. Shown are representative images of spheroids undergoing polarized differentiation, forming a lumen (upper panel); and spheroids with disrupted polarization (lower panel). ( c and d ) BTG3 overexpression disrupted the polarized growth of PC3 cells ( c ), and reduced the plating efficiency in 3D culture ( d ). The mean±S.D. of three independent experiments is shown. ( e–g ) The BTG3-mHW mutant failed to disrupt the polarized growth in 3D culture. Representative microscopic images are shown in ( e ), and expression of the wild-type (WT) and mHW BTG3 proteins in Tet-On PC3 cells, as examined by western blotting, is shown in ( f ). Quantitative results from four independent experiments are shown in ( g ). * P
    Anti Akt Pt308, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RasGRF1 is involved in chemokine and growth factor receptor signaling. (A), Effect of RasGRF1 down-regulation on activation of intracellular signaling in ARMS cells. Phosphorylation of p42/44 MAPK and AKT in RH30-derived cell lines was stimulated for 5 min by SDF-1 (300 ng/ml), I-TAC (100 ng/ml), HGF (100 ng/ml), IGF-II (100 ng/ml), and insulin (10 ng/ml). The experiment was repeated three times with similar results. A representative result is shown. (B), RasGRF1 phosphorylation after stimulation with chemokines and growth factors. RasGRF1 protein phophorylated at Ser929 was detected by western blot analysis after stimulation for 5 min by SDF-1 (300 ng/ml), I-TAC (100 ng/ml), HGF (100 ng/ml), IGF-II (100 ng/ml), and insulin (10 ng/ml). (C), Effect of RasGRF1 down-regulation on Ras activation. A Ras pull-down assay was performed on two RH30-derived cell lines (RH30scr and RH30 RasGRF1-kd). The cells were stimulated for 5 min with SDF-1 (300 ng/ml) or IGF-II (100 ng/ml). Ras-GTP was precipitated by Raf-1 RBD agarose conjugate and detected by Ras antibody clone RAS10 (Millipore). The same antibody was used to detect total Ras protein (p21 H-, K- and N-Ras). Western blots were analyzed by densitometry (right side). The experiment was repeated three times with similar results. A representative result is shown. (D), Effect of RasGRF1 down-regulation on paxillin expression and actin cytoskeleton. Staining of paxillin and actin was performed on RH30scr, RH30 RasGRF1-kd, RH18scr and RH18 RasGRF-kd cell lines. The experiment was repeated three times and representative results are shown.

    Journal: International Journal of Oncology

    Article Title: RasGRF1 regulates proliferation and metastatic behavior of human alveolar rhabdomyosarcomas

    doi: 10.3892/ijo.2012.1536

    Figure Lengend Snippet: RasGRF1 is involved in chemokine and growth factor receptor signaling. (A), Effect of RasGRF1 down-regulation on activation of intracellular signaling in ARMS cells. Phosphorylation of p42/44 MAPK and AKT in RH30-derived cell lines was stimulated for 5 min by SDF-1 (300 ng/ml), I-TAC (100 ng/ml), HGF (100 ng/ml), IGF-II (100 ng/ml), and insulin (10 ng/ml). The experiment was repeated three times with similar results. A representative result is shown. (B), RasGRF1 phosphorylation after stimulation with chemokines and growth factors. RasGRF1 protein phophorylated at Ser929 was detected by western blot analysis after stimulation for 5 min by SDF-1 (300 ng/ml), I-TAC (100 ng/ml), HGF (100 ng/ml), IGF-II (100 ng/ml), and insulin (10 ng/ml). (C), Effect of RasGRF1 down-regulation on Ras activation. A Ras pull-down assay was performed on two RH30-derived cell lines (RH30scr and RH30 RasGRF1-kd). The cells were stimulated for 5 min with SDF-1 (300 ng/ml) or IGF-II (100 ng/ml). Ras-GTP was precipitated by Raf-1 RBD agarose conjugate and detected by Ras antibody clone RAS10 (Millipore). The same antibody was used to detect total Ras protein (p21 H-, K- and N-Ras). Western blots were analyzed by densitometry (right side). The experiment was repeated three times with similar results. A representative result is shown. (D), Effect of RasGRF1 down-regulation on paxillin expression and actin cytoskeleton. Staining of paxillin and actin was performed on RH30scr, RH30 RasGRF1-kd, RH18scr and RH18 RasGRF-kd cell lines. The experiment was repeated three times and representative results are shown.

    Article Snippet: Equal loading in the lanes was evaluated by stripping the blots and reprobing with appropriate mAbs: p42/44 anti-MAPK clone no. 9102 and anti-AKT clone no. 9272 (Santa Cruz Biotechnology).

    Techniques: Activation Assay, Derivative Assay, Western Blot, Pull Down Assay, Expressing, Staining

    Protein expression of p-Akt, p-ERK1/2, APJ receptor in the cellular membrane (APJ-CM) and APJ receptor in the cytoplasm (APJ-CP) in isolated left ventricular myocytes of rats with hypertension and heart failure (H-HF). (A) p-Akt, p-ERK1/2, APJ-CM and APJ-CP were expresson was detected by western blot analysis. (B) The intensity of each band on the blot was quantified by densitometric scanning and all values are expressed as the means ± SD. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Effect of apelin on the cardiac hemodynamics in hypertensive rats with heart failure

    doi: 10.3892/ijmm.2014.1829

    Figure Lengend Snippet: Protein expression of p-Akt, p-ERK1/2, APJ receptor in the cellular membrane (APJ-CM) and APJ receptor in the cytoplasm (APJ-CP) in isolated left ventricular myocytes of rats with hypertension and heart failure (H-HF). (A) p-Akt, p-ERK1/2, APJ-CM and APJ-CP were expresson was detected by western blot analysis. (B) The intensity of each band on the blot was quantified by densitometric scanning and all values are expressed as the means ± SD. * P

    Article Snippet: Rabbit polyclonal anti-APJ antibody, anti-total-Akt (t-Akt), anti-phosphorylation-Akt (p-Akt), anti-total-extracellular signal-regulated kinase 1/2 (t-ERK1/2) antibody and anti-phosphorylated-ERK1/2 (p-ERK1/2) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Isolation, Western Blot

    Protein expression of (A) p-Akt, (B) p-ERK1/2, (C) APJ receptor in the cellular membrane (APJ-CM) and (D) APJ receptor in the cytoplasm (APJ-CP) in isolated left ventricular myocytes of rats with hypertension and heart failure (H-HF) treated with 5% glucose injection (GS) or pyroglutamylated apelin-13 (Pyr-AP13). All values are expressed as the means ± SD. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Effect of apelin on the cardiac hemodynamics in hypertensive rats with heart failure

    doi: 10.3892/ijmm.2014.1829

    Figure Lengend Snippet: Protein expression of (A) p-Akt, (B) p-ERK1/2, (C) APJ receptor in the cellular membrane (APJ-CM) and (D) APJ receptor in the cytoplasm (APJ-CP) in isolated left ventricular myocytes of rats with hypertension and heart failure (H-HF) treated with 5% glucose injection (GS) or pyroglutamylated apelin-13 (Pyr-AP13). All values are expressed as the means ± SD. * P

    Article Snippet: Rabbit polyclonal anti-APJ antibody, anti-total-Akt (t-Akt), anti-phosphorylation-Akt (p-Akt), anti-total-extracellular signal-regulated kinase 1/2 (t-ERK1/2) antibody and anti-phosphorylated-ERK1/2 (p-ERK1/2) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Isolation, Injection

    Mdm20 regulates the phosphorylation status of Akt. A. Western blots for Akt with a focus on phosphorylated Akt on Ser473 and Thr308. HEK293 cells were transfected either with mock, Flag-Mdm20 and mCherry-Nat5 or control, Mdm20 and Nat5 siRNA. At 48 hrs or 72hrs post-transfection, respectively, the cells were harvested, and cell lysates were prepared and processed for western blot analysis. The antibodies used are indicated. For details, see Materials and Methods. B. Western blots were used to determine the phosphorylation status of GSK3β, PTEN, PDK-1, mTOR, and PP1. The antibodies used were as indicated. Note that the phosphorylation of mTOR (Ser-2481) is consistently reduced in Mdm20-KD cells. C. Akt inhibition increases the levels of LC3II. Shown is western blots of HEK293 cellular extracts treated or untreated with siRNAs for Mdm20 or Nat5 in the absence (DMSO control) or presence of Akt inhibitor (Akt-I-VIII). Levels of Akt, and phospho-Akt (pAkt-Ser473), and LC3 levels are shown with the loading control of actin blot.

    Journal: PLoS ONE

    Article Title: Mdm20 Stimulates PolyQ Aggregation via Inhibiting Autophagy Through Akt-Ser473 Phosphorylation

    doi: 10.1371/journal.pone.0082523

    Figure Lengend Snippet: Mdm20 regulates the phosphorylation status of Akt. A. Western blots for Akt with a focus on phosphorylated Akt on Ser473 and Thr308. HEK293 cells were transfected either with mock, Flag-Mdm20 and mCherry-Nat5 or control, Mdm20 and Nat5 siRNA. At 48 hrs or 72hrs post-transfection, respectively, the cells were harvested, and cell lysates were prepared and processed for western blot analysis. The antibodies used are indicated. For details, see Materials and Methods. B. Western blots were used to determine the phosphorylation status of GSK3β, PTEN, PDK-1, mTOR, and PP1. The antibodies used were as indicated. Note that the phosphorylation of mTOR (Ser-2481) is consistently reduced in Mdm20-KD cells. C. Akt inhibition increases the levels of LC3II. Shown is western blots of HEK293 cellular extracts treated or untreated with siRNAs for Mdm20 or Nat5 in the absence (DMSO control) or presence of Akt inhibitor (Akt-I-VIII). Levels of Akt, and phospho-Akt (pAkt-Ser473), and LC3 levels are shown with the loading control of actin blot.

    Article Snippet: The other antibodies used in this study were purchased as follows: anti-Flag, anti-β-actin and anti-MAP2 were purchased from Sigma; anti-ribosomal protein L3, anti-Myc, anti-vimentin and anti-PP1 antibody were purchased from Santa Cruz; anti-phospho-Akt (Ser473 and Thr308), anti-Akt, anti-GSK3β, anti-phospho-GSK3β, anti-mTOR, anti-phospho-mTOR (Ser2481), anti-phospho-PDK (Ser241) were purchased from Cell Signaling Technology; anti-LC3 was purchased from Medical and Biological Laboratories (MBL); anti-ubiquitin was purchased from Millipore; anti-GFP was purchased from Nacalai Tesque; MG132 and 3-methyladenine were purchased from Sigma, ammonium chloride was from Nacalai Tesque, rapamycin was from Cell Signaling Technology and Akt inhibitor VIII was from Merck.

    Techniques: Western Blot, Transfection, Inhibition

    Mdm20 affects the polyQ aggregate formation by the regulation of pAkt Ser473 level. A. A phospho-mimic mutant of Akt (Akt-S473D) increases polyQ aggregate formation. Left: Shown are western blots for Akt and phospho-Akt (at Ser473 and Thr308). HEK293 cells were co-transfected with GFP-polyQ81 and Flag-tagged wild type Akt (F-Akt) or various phospho- and none-phospho-mimic Akt mutants as indicated. Right: The relative levels of polyQ aggregate formation were evaluated in each of the transfectants and compared with the mock-transfected control. Experiments were performed in naïve HEK293 cells (green bars) and Mdm20-KD cells (red bars). The numbers of polyQ-bearing cells among the GFP-positive cells were calculated and normalized to the level of mock transfection. The data represent the mean +/- S.D. (n=3) ***P

    Journal: PLoS ONE

    Article Title: Mdm20 Stimulates PolyQ Aggregation via Inhibiting Autophagy Through Akt-Ser473 Phosphorylation

    doi: 10.1371/journal.pone.0082523

    Figure Lengend Snippet: Mdm20 affects the polyQ aggregate formation by the regulation of pAkt Ser473 level. A. A phospho-mimic mutant of Akt (Akt-S473D) increases polyQ aggregate formation. Left: Shown are western blots for Akt and phospho-Akt (at Ser473 and Thr308). HEK293 cells were co-transfected with GFP-polyQ81 and Flag-tagged wild type Akt (F-Akt) or various phospho- and none-phospho-mimic Akt mutants as indicated. Right: The relative levels of polyQ aggregate formation were evaluated in each of the transfectants and compared with the mock-transfected control. Experiments were performed in naïve HEK293 cells (green bars) and Mdm20-KD cells (red bars). The numbers of polyQ-bearing cells among the GFP-positive cells were calculated and normalized to the level of mock transfection. The data represent the mean +/- S.D. (n=3) ***P

    Article Snippet: The other antibodies used in this study were purchased as follows: anti-Flag, anti-β-actin and anti-MAP2 were purchased from Sigma; anti-ribosomal protein L3, anti-Myc, anti-vimentin and anti-PP1 antibody were purchased from Santa Cruz; anti-phospho-Akt (Ser473 and Thr308), anti-Akt, anti-GSK3β, anti-phospho-GSK3β, anti-mTOR, anti-phospho-mTOR (Ser2481), anti-phospho-PDK (Ser241) were purchased from Cell Signaling Technology; anti-LC3 was purchased from Medical and Biological Laboratories (MBL); anti-ubiquitin was purchased from Millipore; anti-GFP was purchased from Nacalai Tesque; MG132 and 3-methyladenine were purchased from Sigma, ammonium chloride was from Nacalai Tesque, rapamycin was from Cell Signaling Technology and Akt inhibitor VIII was from Merck.

    Techniques: Mutagenesis, Western Blot, Transfection

    BTG3 suppresses growth of PC3 prostate cancer cells in 3D culture. ( a ) Characterization of PC3 Tet-On cells that express myc-tagged BTG3 upon the addition of doxycycline (+). TR, control cells that express only the tetracycline regulator; ovBTG3, Tet-On BTG3-overexpressing cells (clone no. 6). AKT T308 phosphorylation (pT308) was downregulated in ovBTG3 cells. ( b ) Immunofluorescence microscopy of spheroids grown in 3D culture. Shown are representative images of spheroids undergoing polarized differentiation, forming a lumen (upper panel); and spheroids with disrupted polarization (lower panel). ( c and d ) BTG3 overexpression disrupted the polarized growth of PC3 cells ( c ), and reduced the plating efficiency in 3D culture ( d ). The mean±S.D. of three independent experiments is shown. ( e–g ) The BTG3-mHW mutant failed to disrupt the polarized growth in 3D culture. Representative microscopic images are shown in ( e ), and expression of the wild-type (WT) and mHW BTG3 proteins in Tet-On PC3 cells, as examined by western blotting, is shown in ( f ). Quantitative results from four independent experiments are shown in ( g ). * P

    Journal: Cell Death & Disease

    Article Title: Candidate tumor suppressor B-cell translocation gene 3 impedes neoplastic progression by suppression of AKT

    doi: 10.1038/cddis.2014.550

    Figure Lengend Snippet: BTG3 suppresses growth of PC3 prostate cancer cells in 3D culture. ( a ) Characterization of PC3 Tet-On cells that express myc-tagged BTG3 upon the addition of doxycycline (+). TR, control cells that express only the tetracycline regulator; ovBTG3, Tet-On BTG3-overexpressing cells (clone no. 6). AKT T308 phosphorylation (pT308) was downregulated in ovBTG3 cells. ( b ) Immunofluorescence microscopy of spheroids grown in 3D culture. Shown are representative images of spheroids undergoing polarized differentiation, forming a lumen (upper panel); and spheroids with disrupted polarization (lower panel). ( c and d ) BTG3 overexpression disrupted the polarized growth of PC3 cells ( c ), and reduced the plating efficiency in 3D culture ( d ). The mean±S.D. of three independent experiments is shown. ( e–g ) The BTG3-mHW mutant failed to disrupt the polarized growth in 3D culture. Representative microscopic images are shown in ( e ), and expression of the wild-type (WT) and mHW BTG3 proteins in Tet-On PC3 cells, as examined by western blotting, is shown in ( f ). Quantitative results from four independent experiments are shown in ( g ). * P

    Article Snippet: The antibodies used were anti-BTG3 (1 : 150; 4) and anti-AKT pT308 (1 : 250; sc-16646-R; Santa Cruz Biotechnology, Dallas, TX, USA).

    Techniques: Immunofluorescence, Microscopy, Over Expression, Mutagenesis, Expressing, Western Blot