extracellular signal regulated kinase  (Abcam)

 
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    ERK1 2 ELISA Kit
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    ab176641
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    Abcam extracellular signal regulated kinase

    https://www.bioz.com/result/extracellular signal regulated kinase/product/Abcam
    Average 98 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
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    98/100 stars

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    Enzyme-linked Immunosorbent Assay:

    Article Title: Bisphenol a Exposure, DNA Methylation, and Asthma in Children
    Article Snippet: .. Analysis of Plasma Level of MAPK1 Protein For the quantitative measurements of MAPK1 (ERK1/2), an ELISA kit was used on lysed human leukocytes as described (ERK1/2 SimpleStep ELISA Kit, ab176641, Abcam, Cambridge, UK) [ ]. ..

    other:

    Article Title: The Long Noncoding RNA ZFAS1 Potentiates the Development of Hepatocellular Carcinoma via the microRNA-624/MDK/ERK/JNK/P38 Signaling Pathway
    Article Snippet: The antibodies used in this work were JNK (1:5000, ab110724), pJNK (1:5000, ab47337); ERK 1/2 (1:3000, ab176641); pERK 1/2 (1:5000, ab223570), MDK (1:2000, ab52637), E-cadherin (1:5000, ab76055), Vimentin (1:5000, ab92547), GAPDH (1:10,000, ab181602), and horseradish peroxidase-linked goat anti-rabbit secondary antibody against IgG (1:50,000, ab6721).

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  • 90
    Abcam p h3s10
    <t>p-H3S10</t> rapidly increases in the ipsilateral dorsal horn after inflammation was induced through hind paw formalin injection and is expressed predominantly in neurons. (A) Representative images of nuclear p-H3S10 distribution within the L4 dorsal horn 30 minutes after formalin injection. White lines indicate border between gray and white matter. Low magnification: scale bar, 50 μm. High magnification: scale bar, 30 μm. (B) Ipsilateral and contralateral counts of p-H3S10 in laminae I and II and laminae III–V (n = 3 each time point, 5 sections per animal). Data show mean ± SEM (per 40 μm section). * P
    P H3s10, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p h3s10/product/Abcam
    Average 90 stars, based on 1 article reviews
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    ldhb  (Abcam)
    90
    Abcam ldhb
    ATPR regulates the <t>Raf/MEK/ERK</t> signalling pathway through <t>LDHB</t> and the effects of LDHB on tumour growth in vivo (A) Protein expression of Raf, p‐MEK, MEK, p‐ERK and ERK was determined by Western blotting analysis after treatment with ATPR for different concentration (10 −7 ,10 −6 ,10 −5 ) (Mean ± SD, n = 3). (B) Protein expression of Raf, p‐MEK, MEK, p‐ERK and ERK was determined by western blotting analysis after treatment with ATPR in the absence or in the presence of the RARα‐selective antagonist Ro 41‐5253 (Mean ± SD, n = 3). (C) Protein expression of Raf, p‐MEK, MEK, p‐ERK and ERK was determined by Western blotting analysis after LDHB depletion in NB4 and MOLM‐13 cells (Mean ± SD, n = 3). (D) Representative two tumour tissues from vehicle control mice and ATPR‐treated mice group was fixed and immunohistochemistry staining for LDHB. (E, F) Tumour images and weights at experimental endpoints in NC and shLDHB xenografts (Mean ± SD, n = 6). (G) Tumour volumes were measured every day (Mean ± SD, n = 6). (H) Immunohistochemistry staining for LDHB, CD11b and KI67 of NC and shLDHB in NB4 and MOLM‐13 group. * P
    Ldhb, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam npm1 ma
    INPP4B is upregulated by <t>NPM1-mA</t> in leukemia cells via ERK/Ets-1 signaling. ( a ) qRT-PCR analysis of INPP4B mRNA expression, ( b ) western blotting analysis of INPP4B, p-SGK3 T320 , SGK3 and NPM1-mA from the NPM1-mA-silenced OCI-AML3 cells. ( c ) qRT-PCR analysis of INPP4B mRNA expression, ( d ) western blotting analysis of INPP4B, p-SGK3 T320 , SGK3 and NPM1-mA from the THP-1 and K562 cells transduced with the plasmids expressing NPM1-wt or NPM1-mA. ( e ) qRT-PCR analysis of INPP4B mRNA expression, ( f ) western blotting analysis of INPP4B, p-SGK3 T320 , SGK3, p-Ets-1 and Ets-1 from the OCI-AML3 cells transfected with the control siRNA or siEts-1. ( g ) Western blotting analysis of p-ERK, ERK, p-Ets-1, Ets-1, INPP4B, p-SGK3 T320 and SGK3, ( h ) qRT-PCR analysis of INPP4B mRNA expression from the OCI-AML3 cells treated with different concentration of PD98059 (0, 10, 20 and 40 μM). i Western blotting analysis of p-ERK, ERK, p-Ets-1, Ets-1, INPP4B, p-SGK3 T320 , SGK3 and NPM1-mA from the NPM1-mA-silenced OCI-AML3 cells. Proteins were quantified using image software and normalized against β-actin. Data were represented as mean ± s.d. of three individual experiments. * p
    Npm1 Ma, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    akt  (Abcam)
    93
    Abcam akt
    VS-5584 treatment causes activation of <t>ERK</t> in PDAC cells ( A and B ) BxPC-3 and HPAC cells were treated with vehicle control or variable concentrations of VS-5584 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to vehicle control, are indicated. ( C and D ) BxPC-3 and HPAC cells were treated with vehicle control or 2 μM VS-5584 for 4, 8, 12, 24, or 48 h. Whole cell lysates were subjected to Western blotting and probed with <t>anti-p-AKT(S473),</t> -p-AKT(T308), -AKT, -p-S6, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( E and F ) BxPC-3 and HPAC cells were treated with vehicle control or variable concentrations of VS-5584 for 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-ERK, -ERK, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( G and H ) BxPC-3 and HPAC cells were treated with vehicle control or 2 μM VS-5584 for 4, 8, 12, 24, or 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-ERK, -ERK, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated.
    Akt, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    p-H3S10 rapidly increases in the ipsilateral dorsal horn after inflammation was induced through hind paw formalin injection and is expressed predominantly in neurons. (A) Representative images of nuclear p-H3S10 distribution within the L4 dorsal horn 30 minutes after formalin injection. White lines indicate border between gray and white matter. Low magnification: scale bar, 50 μm. High magnification: scale bar, 30 μm. (B) Ipsilateral and contralateral counts of p-H3S10 in laminae I and II and laminae III–V (n = 3 each time point, 5 sections per animal). Data show mean ± SEM (per 40 μm section). * P

    Journal: Pain

    Article Title: The mitogen and stress-activated protein kinase 1 regulates the rapid epigenetic tagging of dorsal horn neurons and nocifensive behaviour

    doi: 10.1097/j.pain.0000000000000679

    Figure Lengend Snippet: p-H3S10 rapidly increases in the ipsilateral dorsal horn after inflammation was induced through hind paw formalin injection and is expressed predominantly in neurons. (A) Representative images of nuclear p-H3S10 distribution within the L4 dorsal horn 30 minutes after formalin injection. White lines indicate border between gray and white matter. Low magnification: scale bar, 50 μm. High magnification: scale bar, 30 μm. (B) Ipsilateral and contralateral counts of p-H3S10 in laminae I and II and laminae III–V (n = 3 each time point, 5 sections per animal). Data show mean ± SEM (per 40 μm section). * P

    Article Snippet: In particular, the MEK inhibitor SL327 has been shown to attenuate both the first and second phase of the formalin response. p-MSK1 is a well-characterised kinase directly downstream of p-ERK which can induce p-H3S10 (Fig. A).

    Techniques: Injection

    Spinal extracellular signal-regulated kinase (ERK) regulates p-MSK1 and p-H3S10 expression after formalin injection. (A) Diagram of postulated signaling pathways upstream of p-H3S10. (B) Dorsal horn images of p-ERK and p-MSK1 in vehicle- and MEK inhibitor SL327-treated animals 30 minutes after formalin stimulation. (C) Dorsal horn images of p-ERK and p-H3S10 in vehicle- and MEK inhibitor SL327-treated animals 30 minutes after formalin stimulation. (D) Dorsal horn images of p-H3S10 and p-MSK1 in vehicle- and MEK inhibitor SL327-treated animals 30 minutes after formalin stimulation. (B-D): Pictures show a single focal plane. Final column images show a merge of the first 2 images; colocalisation is seen in yellow; arrowheads indicate examples of colocalisation. The white line indicates the medial border between lamina I and white matter. Scale bar, 50 μm. (E) Quantification of p-ERK, p-H3S10, and p-MSK1 single-labeled cells 30 minutes after formalin injection. n = 8 in each treatment group, 5 sections per animal. (F) Quantification of p-ERK, p-H3S10, and p-MSK1 double-labeled cells 30 minutes after formalin. n = 4 in each group. (E-F) Values presented as group mean ± SEM (per 40 μm section). * P

    Journal: Pain

    Article Title: The mitogen and stress-activated protein kinase 1 regulates the rapid epigenetic tagging of dorsal horn neurons and nocifensive behaviour

    doi: 10.1097/j.pain.0000000000000679

    Figure Lengend Snippet: Spinal extracellular signal-regulated kinase (ERK) regulates p-MSK1 and p-H3S10 expression after formalin injection. (A) Diagram of postulated signaling pathways upstream of p-H3S10. (B) Dorsal horn images of p-ERK and p-MSK1 in vehicle- and MEK inhibitor SL327-treated animals 30 minutes after formalin stimulation. (C) Dorsal horn images of p-ERK and p-H3S10 in vehicle- and MEK inhibitor SL327-treated animals 30 minutes after formalin stimulation. (D) Dorsal horn images of p-H3S10 and p-MSK1 in vehicle- and MEK inhibitor SL327-treated animals 30 minutes after formalin stimulation. (B-D): Pictures show a single focal plane. Final column images show a merge of the first 2 images; colocalisation is seen in yellow; arrowheads indicate examples of colocalisation. The white line indicates the medial border between lamina I and white matter. Scale bar, 50 μm. (E) Quantification of p-ERK, p-H3S10, and p-MSK1 single-labeled cells 30 minutes after formalin injection. n = 8 in each treatment group, 5 sections per animal. (F) Quantification of p-ERK, p-H3S10, and p-MSK1 double-labeled cells 30 minutes after formalin. n = 4 in each group. (E-F) Values presented as group mean ± SEM (per 40 μm section). * P

    Article Snippet: In particular, the MEK inhibitor SL327 has been shown to attenuate both the first and second phase of the formalin response. p-MSK1 is a well-characterised kinase directly downstream of p-ERK which can induce p-H3S10 (Fig. A).

    Techniques: Expressing, Injection, Labeling

    Inhibition of MSK1 with SB747651A (10 μM) causes a reduction in formalin-induced p-H3S10 but has no effect on p-ERK and p-MSK1 expression. (A) Images of spinal p-H3S10 in vehicle- and SB747651A (10 μM)-treated animals, 1 hour after formalin injection. Scale bar, 100 μm; the white line indicates the medial border of lamina I and white matter. (B) Quantification of p-H3S10, p-ERK, and p-MSK1 in the dorsal horn of the vehicle or SB747651A (10 μM) formalin-treated animals (n = 8 in each group, 5 sections per animal). Values presented as group mean ± SEM (per 40 μm section).

    Journal: Pain

    Article Title: The mitogen and stress-activated protein kinase 1 regulates the rapid epigenetic tagging of dorsal horn neurons and nocifensive behaviour

    doi: 10.1097/j.pain.0000000000000679

    Figure Lengend Snippet: Inhibition of MSK1 with SB747651A (10 μM) causes a reduction in formalin-induced p-H3S10 but has no effect on p-ERK and p-MSK1 expression. (A) Images of spinal p-H3S10 in vehicle- and SB747651A (10 μM)-treated animals, 1 hour after formalin injection. Scale bar, 100 μm; the white line indicates the medial border of lamina I and white matter. (B) Quantification of p-H3S10, p-ERK, and p-MSK1 in the dorsal horn of the vehicle or SB747651A (10 μM) formalin-treated animals (n = 8 in each group, 5 sections per animal). Values presented as group mean ± SEM (per 40 μm section).

    Article Snippet: In particular, the MEK inhibitor SL327 has been shown to attenuate both the first and second phase of the formalin response. p-MSK1 is a well-characterised kinase directly downstream of p-ERK which can induce p-H3S10 (Fig. A).

    Techniques: Inhibition, Expressing, Injection

    Depletion of spinal serotonin prevents the full expression of formalin-induced p-H3S10. (A) Typical dorsal horn image of 5-HT and p-H3S10 staining in animals receiving i.t. saline or i.t. (5,7-DHT), 30 minutes after formalin stimulation. Scale bar, 50 μm (upper), 30 μm (lower). (B) Counts of p-H3S10 nuclei in the ipsilateral and contralateral dorsal horn (n = 7 each group, 5 sections per animal). Data show group mean ± SEM (per 40-μm section). ** P

    Journal: Pain

    Article Title: The mitogen and stress-activated protein kinase 1 regulates the rapid epigenetic tagging of dorsal horn neurons and nocifensive behaviour

    doi: 10.1097/j.pain.0000000000000679

    Figure Lengend Snippet: Depletion of spinal serotonin prevents the full expression of formalin-induced p-H3S10. (A) Typical dorsal horn image of 5-HT and p-H3S10 staining in animals receiving i.t. saline or i.t. (5,7-DHT), 30 minutes after formalin stimulation. Scale bar, 50 μm (upper), 30 μm (lower). (B) Counts of p-H3S10 nuclei in the ipsilateral and contralateral dorsal horn (n = 7 each group, 5 sections per animal). Data show group mean ± SEM (per 40-μm section). ** P

    Article Snippet: In particular, the MEK inhibitor SL327 has been shown to attenuate both the first and second phase of the formalin response. p-MSK1 is a well-characterised kinase directly downstream of p-ERK which can induce p-H3S10 (Fig. A).

    Techniques: Expressing, Staining

    p-H3S10 expression occurs within neurons of the pain pathways. (A) Dorsal horn images of p-ERK (green) and p-H3S10 (red) double labelling in laminae I and II. In the merge, colocalisation is seen in yellow. Scale bar, 30 μm. (B) Dorsal horn images of c-Fos (green) and p-H3S10 (red) double labeling in formalin-stimulated animals. In the merge, colocalisation is seen in yellow. Scale bar, 50 μm. (C) Dorsal horn images of Zif268 (green) and p-H3S10 (red) double labeling. In the merge, colocalisation is seen in yellow. Scale bar, 50 μm. (A-D) White lines indicate medial border between lamina I and white matter; arrowheads indicate examples of colocalisation. All sections are 40 μm shown taken in a single focal plane in a formalin-stimulated animal. (D) Dorsal horn images of NK1R (green) and p-H3S10 (red) double labeling. The final image shows a merge of the first 2 images; NK1R is a cell-surface receptor and thus colocalisation is shown as NK1R surrounding the p-H3S10-labelled nucleus. Scale bar, 30 μm.

    Journal: Pain

    Article Title: The mitogen and stress-activated protein kinase 1 regulates the rapid epigenetic tagging of dorsal horn neurons and nocifensive behaviour

    doi: 10.1097/j.pain.0000000000000679

    Figure Lengend Snippet: p-H3S10 expression occurs within neurons of the pain pathways. (A) Dorsal horn images of p-ERK (green) and p-H3S10 (red) double labelling in laminae I and II. In the merge, colocalisation is seen in yellow. Scale bar, 30 μm. (B) Dorsal horn images of c-Fos (green) and p-H3S10 (red) double labeling in formalin-stimulated animals. In the merge, colocalisation is seen in yellow. Scale bar, 50 μm. (C) Dorsal horn images of Zif268 (green) and p-H3S10 (red) double labeling. In the merge, colocalisation is seen in yellow. Scale bar, 50 μm. (A-D) White lines indicate medial border between lamina I and white matter; arrowheads indicate examples of colocalisation. All sections are 40 μm shown taken in a single focal plane in a formalin-stimulated animal. (D) Dorsal horn images of NK1R (green) and p-H3S10 (red) double labeling. The final image shows a merge of the first 2 images; NK1R is a cell-surface receptor and thus colocalisation is shown as NK1R surrounding the p-H3S10-labelled nucleus. Scale bar, 30 μm.

    Article Snippet: In particular, the MEK inhibitor SL327 has been shown to attenuate both the first and second phase of the formalin response. p-MSK1 is a well-characterised kinase directly downstream of p-ERK which can induce p-H3S10 (Fig. A).

    Techniques: Expressing, Labeling, Cell Surface Receptor Assay

    ATPR regulates the Raf/MEK/ERK signalling pathway through LDHB and the effects of LDHB on tumour growth in vivo (A) Protein expression of Raf, p‐MEK, MEK, p‐ERK and ERK was determined by Western blotting analysis after treatment with ATPR for different concentration (10 −7 ,10 −6 ,10 −5 ) (Mean ± SD, n = 3). (B) Protein expression of Raf, p‐MEK, MEK, p‐ERK and ERK was determined by western blotting analysis after treatment with ATPR in the absence or in the presence of the RARα‐selective antagonist Ro 41‐5253 (Mean ± SD, n = 3). (C) Protein expression of Raf, p‐MEK, MEK, p‐ERK and ERK was determined by Western blotting analysis after LDHB depletion in NB4 and MOLM‐13 cells (Mean ± SD, n = 3). (D) Representative two tumour tissues from vehicle control mice and ATPR‐treated mice group was fixed and immunohistochemistry staining for LDHB. (E, F) Tumour images and weights at experimental endpoints in NC and shLDHB xenografts (Mean ± SD, n = 6). (G) Tumour volumes were measured every day (Mean ± SD, n = 6). (H) Immunohistochemistry staining for LDHB, CD11b and KI67 of NC and shLDHB in NB4 and MOLM‐13 group. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: ATPR triggers acute myeloid leukaemia cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis, et al. ATPR triggers acute myeloid leukaemia cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis

    doi: 10.1111/jcmm.15353

    Figure Lengend Snippet: ATPR regulates the Raf/MEK/ERK signalling pathway through LDHB and the effects of LDHB on tumour growth in vivo (A) Protein expression of Raf, p‐MEK, MEK, p‐ERK and ERK was determined by Western blotting analysis after treatment with ATPR for different concentration (10 −7 ,10 −6 ,10 −5 ) (Mean ± SD, n = 3). (B) Protein expression of Raf, p‐MEK, MEK, p‐ERK and ERK was determined by western blotting analysis after treatment with ATPR in the absence or in the presence of the RARα‐selective antagonist Ro 41‐5253 (Mean ± SD, n = 3). (C) Protein expression of Raf, p‐MEK, MEK, p‐ERK and ERK was determined by Western blotting analysis after LDHB depletion in NB4 and MOLM‐13 cells (Mean ± SD, n = 3). (D) Representative two tumour tissues from vehicle control mice and ATPR‐treated mice group was fixed and immunohistochemistry staining for LDHB. (E, F) Tumour images and weights at experimental endpoints in NC and shLDHB xenografts (Mean ± SD, n = 6). (G) Tumour volumes were measured every day (Mean ± SD, n = 6). (H) Immunohistochemistry staining for LDHB, CD11b and KI67 of NC and shLDHB in NB4 and MOLM‐13 group. * P

    Article Snippet: Antibodies against human PU.1, Cyclin A2, Cyclin D3, CDK4, p‐MEK, MEK, Raf, p‐ERK 1/2, ERK 1/2 and LDHB (Abcam) were diluted at 1:2000 dilution and anti‐β‐actin (ZSGB‐BIO, China) was used at 1:300.

    Techniques: In Vivo, Expressing, Western Blot, Concentration Assay, Mouse Assay, Immunohistochemistry, Staining

    ATPR triggers AML cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis. ATPR triggers AML cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: ATPR triggers acute myeloid leukaemia cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis, et al. ATPR triggers acute myeloid leukaemia cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis

    doi: 10.1111/jcmm.15353

    Figure Lengend Snippet: ATPR triggers AML cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis. ATPR triggers AML cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis

    Article Snippet: Antibodies against human PU.1, Cyclin A2, Cyclin D3, CDK4, p‐MEK, MEK, Raf, p‐ERK 1/2, ERK 1/2 and LDHB (Abcam) were diluted at 1:2000 dilution and anti‐β‐actin (ZSGB‐BIO, China) was used at 1:300.

    Techniques:

    Knockdown LDHB expression inhibits proliferation while promoting differentiation and glycolysis of AML cells. (A) Western analysis of LDHB expression in AML patients PBMC compared with normal human PBMC (Mean ± SD, n = 3). (B) Western analysis of LDHB expression in AML cell lines (Mean ± SD, n = 3). (C, D) After treatment with ATPR (0, 10 −9 , 10 −8 , 10 −7 , 10 −6 , or 10 −5 M) for different durations (24–72 h). The protein expression of LDHB was assessed by Western blotting (Mean ± SD, n = 3). (E) NB4 and MOLM‐13 cells were treated with ATPR in the absence or in the presence of the RARα‐selective antagonist Ro 41‐5253. The protein expression of LDHB was assessed by Western blotting (Mean ± SD, n = 3). (F) Stable control and shLDHB‐transfected NB4 cells were observed by inversed fluorescent microscope. (G) The protein expression of LDHB was assessed by Western blotting after treatment with shLDHB for 7 days (Mean ± SD, n = 3). (H) Cell growth of NB4 and MPOLM‐13 cells after transfection with shLDHB as determined by the CCK‐8 assay at different time points (Mean ± SD, n = 3). (I) The cell cycle distribution was analysed by flow cytometry in NB4 and MOLM‐13 cells after LDHB depletion (Mean ± SD, n = 3). (J) Immunofluorescence was used to detect KI67 in NB4 and MOLM‐13 cells after LDHB depletion. (K) After LDHB depletion, NB4 and MOLM‐13 cells were stained with Wright‐Giemsa dye and the cell morphological features were observed under a microscopy. (L) NBT reduction experiment was performed to count the positive cell rate (Mean ± SD, n = 3). (M) CD11b and CD14 expression were analysed by flow cytometer in NB4 and MOLM‐13 cells after LDHB depletion (Mean ± SD, n = 3). (N) The protein expression of PU.1, Cyclin A2, CDK4 and Cyclin D3 was assessed by Western blotting after LDHB depletion (Mean ± SD, n = 3). * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: ATPR triggers acute myeloid leukaemia cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis, et al. ATPR triggers acute myeloid leukaemia cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis

    doi: 10.1111/jcmm.15353

    Figure Lengend Snippet: Knockdown LDHB expression inhibits proliferation while promoting differentiation and glycolysis of AML cells. (A) Western analysis of LDHB expression in AML patients PBMC compared with normal human PBMC (Mean ± SD, n = 3). (B) Western analysis of LDHB expression in AML cell lines (Mean ± SD, n = 3). (C, D) After treatment with ATPR (0, 10 −9 , 10 −8 , 10 −7 , 10 −6 , or 10 −5 M) for different durations (24–72 h). The protein expression of LDHB was assessed by Western blotting (Mean ± SD, n = 3). (E) NB4 and MOLM‐13 cells were treated with ATPR in the absence or in the presence of the RARα‐selective antagonist Ro 41‐5253. The protein expression of LDHB was assessed by Western blotting (Mean ± SD, n = 3). (F) Stable control and shLDHB‐transfected NB4 cells were observed by inversed fluorescent microscope. (G) The protein expression of LDHB was assessed by Western blotting after treatment with shLDHB for 7 days (Mean ± SD, n = 3). (H) Cell growth of NB4 and MPOLM‐13 cells after transfection with shLDHB as determined by the CCK‐8 assay at different time points (Mean ± SD, n = 3). (I) The cell cycle distribution was analysed by flow cytometry in NB4 and MOLM‐13 cells after LDHB depletion (Mean ± SD, n = 3). (J) Immunofluorescence was used to detect KI67 in NB4 and MOLM‐13 cells after LDHB depletion. (K) After LDHB depletion, NB4 and MOLM‐13 cells were stained with Wright‐Giemsa dye and the cell morphological features were observed under a microscopy. (L) NBT reduction experiment was performed to count the positive cell rate (Mean ± SD, n = 3). (M) CD11b and CD14 expression were analysed by flow cytometer in NB4 and MOLM‐13 cells after LDHB depletion (Mean ± SD, n = 3). (N) The protein expression of PU.1, Cyclin A2, CDK4 and Cyclin D3 was assessed by Western blotting after LDHB depletion (Mean ± SD, n = 3). * P

    Article Snippet: Antibodies against human PU.1, Cyclin A2, Cyclin D3, CDK4, p‐MEK, MEK, Raf, p‐ERK 1/2, ERK 1/2 and LDHB (Abcam) were diluted at 1:2000 dilution and anti‐β‐actin (ZSGB‐BIO, China) was used at 1:300.

    Techniques: Expressing, Western Blot, Transfection, Microscopy, CCK-8 Assay, Flow Cytometry, Immunofluorescence, Staining

    ATPR shows antileukaemic effects with RARα dependent. (A) The NB4 cells were treated with ATPR (10 −6 M) for different durations (24h–72 h). The protein expression of PML‐RARα, RARα, RARβ and RARγ was assessed by Western blotting (Mean ± SD, n = 3). (B) Values are presented as the mean ± SD (n = 3) of three in Quantitative real‐time PCR analysis of mRNA expression of RARα, RARβ, RARγ, CRABP2 and CYP26A1 treated with ATPR (10 −6 M) for 48h in NB4 cells‐dependent experiments. (C) The MOLM‐13 cells were treated with ATPR (10 −6 M) for different durations (24h–72h). The protein expression of RARα, RARβ and RARγ was assessed by Western blotting (Mean ± SD, n = 3). (D) Values are presented as the mean ± SD of three in Quantitative real‐time PCR analysis of mRNA expression of RARα, RARβ, RARγ, CRABP2 and CYP26A1 treated with ATPR (10 −6 M) for 48h in MOLM‐13 cells‐dependent experiments. (E) The cell cycle distribution was analysed by flow cytometry (Mean ± SD, n = 3). (F) CD11b expression was analysed by flow cytometer. (G) The protein expression of PU.1, Cyclin A2, CDK4 and Cyclin D3 was assessed by Western blotting (Mean ± SD, n = 3). (H) AML cells were exposed to various concentrations (0, 2, 4, 8 or 10mM) of 2‐DG for 72 h, followed by the determination of cell viability using the CCK8 assay. (I) AML cells were exposed to various concentrations (0, 10 −7 , 10 −6 , or 10 −5 M) of ATPR for 72 h, followed by the determination of the glycolysis rate using the glucose, lactic acid and ATP detection kit (Mean ± SD, n = 3). (J) Values are presented as the mean ± SD of three in Quantitative real‐time PCR analysis of mRNA expression of LDHB, LDHA, HK2, ENO1 and GAPDH treated with ATPR (10 −6 M) for 48h in MOLM‐13 and NB4 cells‐dependent experiments (K) The OCR was determined in MOLM‐13 and NB4 cells by Seahorse XF. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: ATPR triggers acute myeloid leukaemia cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis, et al. ATPR triggers acute myeloid leukaemia cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis

    doi: 10.1111/jcmm.15353

    Figure Lengend Snippet: ATPR shows antileukaemic effects with RARα dependent. (A) The NB4 cells were treated with ATPR (10 −6 M) for different durations (24h–72 h). The protein expression of PML‐RARα, RARα, RARβ and RARγ was assessed by Western blotting (Mean ± SD, n = 3). (B) Values are presented as the mean ± SD (n = 3) of three in Quantitative real‐time PCR analysis of mRNA expression of RARα, RARβ, RARγ, CRABP2 and CYP26A1 treated with ATPR (10 −6 M) for 48h in NB4 cells‐dependent experiments. (C) The MOLM‐13 cells were treated with ATPR (10 −6 M) for different durations (24h–72h). The protein expression of RARα, RARβ and RARγ was assessed by Western blotting (Mean ± SD, n = 3). (D) Values are presented as the mean ± SD of three in Quantitative real‐time PCR analysis of mRNA expression of RARα, RARβ, RARγ, CRABP2 and CYP26A1 treated with ATPR (10 −6 M) for 48h in MOLM‐13 cells‐dependent experiments. (E) The cell cycle distribution was analysed by flow cytometry (Mean ± SD, n = 3). (F) CD11b expression was analysed by flow cytometer. (G) The protein expression of PU.1, Cyclin A2, CDK4 and Cyclin D3 was assessed by Western blotting (Mean ± SD, n = 3). (H) AML cells were exposed to various concentrations (0, 2, 4, 8 or 10mM) of 2‐DG for 72 h, followed by the determination of cell viability using the CCK8 assay. (I) AML cells were exposed to various concentrations (0, 10 −7 , 10 −6 , or 10 −5 M) of ATPR for 72 h, followed by the determination of the glycolysis rate using the glucose, lactic acid and ATP detection kit (Mean ± SD, n = 3). (J) Values are presented as the mean ± SD of three in Quantitative real‐time PCR analysis of mRNA expression of LDHB, LDHA, HK2, ENO1 and GAPDH treated with ATPR (10 −6 M) for 48h in MOLM‐13 and NB4 cells‐dependent experiments (K) The OCR was determined in MOLM‐13 and NB4 cells by Seahorse XF. * P

    Article Snippet: Antibodies against human PU.1, Cyclin A2, Cyclin D3, CDK4, p‐MEK, MEK, Raf, p‐ERK 1/2, ERK 1/2 and LDHB (Abcam) were diluted at 1:2000 dilution and anti‐β‐actin (ZSGB‐BIO, China) was used at 1:300.

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Flow Cytometry, CCK-8 Assay

    INPP4B is upregulated by NPM1-mA in leukemia cells via ERK/Ets-1 signaling. ( a ) qRT-PCR analysis of INPP4B mRNA expression, ( b ) western blotting analysis of INPP4B, p-SGK3 T320 , SGK3 and NPM1-mA from the NPM1-mA-silenced OCI-AML3 cells. ( c ) qRT-PCR analysis of INPP4B mRNA expression, ( d ) western blotting analysis of INPP4B, p-SGK3 T320 , SGK3 and NPM1-mA from the THP-1 and K562 cells transduced with the plasmids expressing NPM1-wt or NPM1-mA. ( e ) qRT-PCR analysis of INPP4B mRNA expression, ( f ) western blotting analysis of INPP4B, p-SGK3 T320 , SGK3, p-Ets-1 and Ets-1 from the OCI-AML3 cells transfected with the control siRNA or siEts-1. ( g ) Western blotting analysis of p-ERK, ERK, p-Ets-1, Ets-1, INPP4B, p-SGK3 T320 and SGK3, ( h ) qRT-PCR analysis of INPP4B mRNA expression from the OCI-AML3 cells treated with different concentration of PD98059 (0, 10, 20 and 40 μM). i Western blotting analysis of p-ERK, ERK, p-Ets-1, Ets-1, INPP4B, p-SGK3 T320 , SGK3 and NPM1-mA from the NPM1-mA-silenced OCI-AML3 cells. Proteins were quantified using image software and normalized against β-actin. Data were represented as mean ± s.d. of three individual experiments. * p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: INPP4B promotes cell survival via SGK3 activation in NPM1-mutated leukemia

    doi: 10.1186/s13046-018-0675-9

    Figure Lengend Snippet: INPP4B is upregulated by NPM1-mA in leukemia cells via ERK/Ets-1 signaling. ( a ) qRT-PCR analysis of INPP4B mRNA expression, ( b ) western blotting analysis of INPP4B, p-SGK3 T320 , SGK3 and NPM1-mA from the NPM1-mA-silenced OCI-AML3 cells. ( c ) qRT-PCR analysis of INPP4B mRNA expression, ( d ) western blotting analysis of INPP4B, p-SGK3 T320 , SGK3 and NPM1-mA from the THP-1 and K562 cells transduced with the plasmids expressing NPM1-wt or NPM1-mA. ( e ) qRT-PCR analysis of INPP4B mRNA expression, ( f ) western blotting analysis of INPP4B, p-SGK3 T320 , SGK3, p-Ets-1 and Ets-1 from the OCI-AML3 cells transfected with the control siRNA or siEts-1. ( g ) Western blotting analysis of p-ERK, ERK, p-Ets-1, Ets-1, INPP4B, p-SGK3 T320 and SGK3, ( h ) qRT-PCR analysis of INPP4B mRNA expression from the OCI-AML3 cells treated with different concentration of PD98059 (0, 10, 20 and 40 μM). i Western blotting analysis of p-ERK, ERK, p-Ets-1, Ets-1, INPP4B, p-SGK3 T320 , SGK3 and NPM1-mA from the NPM1-mA-silenced OCI-AML3 cells. Proteins were quantified using image software and normalized against β-actin. Data were represented as mean ± s.d. of three individual experiments. * p

    Article Snippet: Moreover, our previous study has verified that ERK signaling is continuously activated by NPM1-mA [ ].

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

    NPM1-mA-mediated INPP4B upregulation promotes cell proliferation in OCI-AML3 cells. The NPM1-mA-silenced OCI-AML3 cells were subjected to ( a ) CCK8 assays and ( b ) colony forming assays. c The NPM1-mA-silenced OCI-AML3 cells were transfected with the pEAK-Flag/INPP4B plasmids, western blotting analysis of INPP4B and NPM1-mA. Proteins were quantified using image software and normalized against β-actin. d CCK-8 assay analysis of cell proliferation in NPM1-mA-silenced OCI-AML3 cells, followed by Flag-INPP4B introduction. Data were represented as mean ± s.d. of three individual experiments. * p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: INPP4B promotes cell survival via SGK3 activation in NPM1-mutated leukemia

    doi: 10.1186/s13046-018-0675-9

    Figure Lengend Snippet: NPM1-mA-mediated INPP4B upregulation promotes cell proliferation in OCI-AML3 cells. The NPM1-mA-silenced OCI-AML3 cells were subjected to ( a ) CCK8 assays and ( b ) colony forming assays. c The NPM1-mA-silenced OCI-AML3 cells were transfected with the pEAK-Flag/INPP4B plasmids, western blotting analysis of INPP4B and NPM1-mA. Proteins were quantified using image software and normalized against β-actin. d CCK-8 assay analysis of cell proliferation in NPM1-mA-silenced OCI-AML3 cells, followed by Flag-INPP4B introduction. Data were represented as mean ± s.d. of three individual experiments. * p

    Article Snippet: Moreover, our previous study has verified that ERK signaling is continuously activated by NPM1-mA [ ].

    Techniques: Transfection, Western Blot, Software, CCK-8 Assay

    High expression of INPP4B is associated with poor survival outcome in NPM1-mutated leukemia. Kaplan-Meier survival data of 153 AML patients were used to analysis ( a ) OS and ( b ) EFS curves according to INPP4B levels. c Heatmap of 38 primary AML samples with NPM1 mutation from TCGA dataset in which INPP4B expression was aligned with patient event (Survival or Death). Kaplan-Meier survival data of patients with NPM1-mutated AML were used to analysis ( d ) OS and ( e ) EFS curves according to INPP4B levels. f Schematic diagram describing the functional significance of INPP4B in the NPM1-mutated leukemia cells. INPP4B promotes leukemia cell survival in a SGK3-dependent and AKT-independent manner. The expression of INPP4B partially upregulated by NPM1-mA is due to ERK/Ets-1 signaling

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: INPP4B promotes cell survival via SGK3 activation in NPM1-mutated leukemia

    doi: 10.1186/s13046-018-0675-9

    Figure Lengend Snippet: High expression of INPP4B is associated with poor survival outcome in NPM1-mutated leukemia. Kaplan-Meier survival data of 153 AML patients were used to analysis ( a ) OS and ( b ) EFS curves according to INPP4B levels. c Heatmap of 38 primary AML samples with NPM1 mutation from TCGA dataset in which INPP4B expression was aligned with patient event (Survival or Death). Kaplan-Meier survival data of patients with NPM1-mutated AML were used to analysis ( d ) OS and ( e ) EFS curves according to INPP4B levels. f Schematic diagram describing the functional significance of INPP4B in the NPM1-mutated leukemia cells. INPP4B promotes leukemia cell survival in a SGK3-dependent and AKT-independent manner. The expression of INPP4B partially upregulated by NPM1-mA is due to ERK/Ets-1 signaling

    Article Snippet: Moreover, our previous study has verified that ERK signaling is continuously activated by NPM1-mA [ ].

    Techniques: Expressing, Mutagenesis, Functional Assay

    High expression levels of INPP4B in leukemia cells with the NPM1 mutation. a RNA-seq mRNA expression data from the TCGA database were used to compare INPP4B expression between AML patients with ( n = 41) and without NPM1 mutation ( n = 130). * p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: INPP4B promotes cell survival via SGK3 activation in NPM1-mutated leukemia

    doi: 10.1186/s13046-018-0675-9

    Figure Lengend Snippet: High expression levels of INPP4B in leukemia cells with the NPM1 mutation. a RNA-seq mRNA expression data from the TCGA database were used to compare INPP4B expression between AML patients with ( n = 41) and without NPM1 mutation ( n = 130). * p

    Article Snippet: Moreover, our previous study has verified that ERK signaling is continuously activated by NPM1-mA [ ].

    Techniques: Expressing, Mutagenesis, RNA Sequencing Assay

    VS-5584 treatment causes activation of ERK in PDAC cells ( A and B ) BxPC-3 and HPAC cells were treated with vehicle control or variable concentrations of VS-5584 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to vehicle control, are indicated. ( C and D ) BxPC-3 and HPAC cells were treated with vehicle control or 2 μM VS-5584 for 4, 8, 12, 24, or 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-AKT(S473), -p-AKT(T308), -AKT, -p-S6, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( E and F ) BxPC-3 and HPAC cells were treated with vehicle control or variable concentrations of VS-5584 for 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-ERK, -ERK, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( G and H ) BxPC-3 and HPAC cells were treated with vehicle control or 2 μM VS-5584 for 4, 8, 12, 24, or 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-ERK, -ERK, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated.

    Journal: Oncotarget

    Article Title: Targeting ERK enhances the cytotoxic effect of the novel PI3K and mTOR dual inhibitor VS-5584 in preclinical models of pancreatic cancer

    doi: 10.18632/oncotarget.17869

    Figure Lengend Snippet: VS-5584 treatment causes activation of ERK in PDAC cells ( A and B ) BxPC-3 and HPAC cells were treated with vehicle control or variable concentrations of VS-5584 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to vehicle control, are indicated. ( C and D ) BxPC-3 and HPAC cells were treated with vehicle control or 2 μM VS-5584 for 4, 8, 12, 24, or 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-AKT(S473), -p-AKT(T308), -AKT, -p-S6, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( E and F ) BxPC-3 and HPAC cells were treated with vehicle control or variable concentrations of VS-5584 for 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-ERK, -ERK, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( G and H ) BxPC-3 and HPAC cells were treated with vehicle control or 2 μM VS-5584 for 4, 8, 12, 24, or 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-ERK, -ERK, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated.

    Article Snippet: Whole cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Inc., Rockford, IL, USA) and immunoblotted with anti-PARP, -Mcl-1, -Bcl-2, -Bcl-xL, -Bax, -actin, -Bad, -ERK (Proteintech, Chicago, IL, USA), -p-AKT (T308), -p-AKT (S473) (Affinity Biosciences, Changzhou, Jiangsu Province, China), -Bim, -pS6 (Cell Signaling Technologies, Danvers, MA, USA), -p-ERK, or -AKT (Abcam, Cambridge, MA, USA), as previously described [ , , , ].

    Techniques: Activation Assay, Western Blot