anti-β-actin antibody Search Results


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  • 99
    Millipore β actin
    Fyn regulates IP 3 -mediated calcium responses. (A) WEHI 7.2 T cells were transfected with Fyn siRNAs (or non-targeting control siRNAs) as described in materials and methods. Fyn levels were measured by western blotting 24 hours post-transfection. <t>β-actin</t>
    β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 67330 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Santa Cruz Biotechnology β actin
    Knockdown of AQP5 suppressed the ability of migration and invasion in HCT116 and SW480 cells. a The ability of migration was measured by scratch wound healing assay after knockdown of AQP5, the migration rate was determined. b The invasiveness of HCT116 and SW480 cells with and without AQP5 silencing was evaluated by transwell assay, invasive cells were counted and expressed as fold change compared to the Mock group. c The expression levels of MMP-2 and MMP-9 were detected by immunoblotting after knockdown of AQP5, the bands were semi-quantified by densitometry, normalized to <t>β-actin</t> expression and expressed as fold change compared with the Mock group. d The activities of MMP-2 and MMP-9 in AQP5-silenced and control cells were assessed by gelatin zymography. Data were presented as the mean ± SD. * p
    β Actin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 50832 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti beta actin antibody
    Single cell traction force measurement in multiple cell types using the protein micropillar arrays. ( A–I ) Fluorescence/Immunofluorescence staining of ( A–C ) bovine nucleus pulposus cells (bNPCs), ( D–F ) human mesenchymal stem cells (hMSCs), ( G–I ) human dermal fibroblasts (hDFs) and ( J–L ) rabbit chondrocyte, which were cultured on the protein micropillar arrays. Green: Immunofluorescence staining of <t>beta-actin;</t> Blue: DAPI showing the nuclei; Red: Rose Bengal showing the cross-sections of the protein micropillars. The micropillars all have a diameter of 1 μm and a reduced elastic modulus of 30 kPa, but are of different heights (i.e., 6 μm, 8 μm and 10 μm), which are equivalent to a stiffness of ( A,D,G,J ) 20.44 nN/μm, ( B,E,H,K ) 8.62 nN/μm, and ( C,F,I,L ) 4.42 nN/μm, respectively. Scale bars, 10 μm. ( M ) Bar chart showing the single cell total traction force of different types of cells cultured on protein micropillar arrays of different stiffness: bovine nucleus pulposus cells (bNPCs, P2 ~ P4); human mesenchymal stem cells (hMSCs, P3 ~ P5); human dermal fibroblasts (hDF, P5 ~ P15) and rabbit chondrocytes (rChondrocytes, P2 ~ P4) cultured for 3 days on protein micropillars (n = 10).
    Monoclonal Anti Beta Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33074 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Cell Signaling Technology Inc β actin
    Loss of Ei24 impairs glucose homeostasis. A , expression of Ei24 in islets from ob/ob mice, GK rats at 4 months of age, and C57BL/6 mice fed an HFD for 2 weeks and 8 weeks. <t>β-Actin</t> served as the loading control. CD , chow diet. B , total RNA was prepared from islets of WT and KO mice at 8 weeks of age. The transcription levels of Ei24 mRNA are normalized to β-actin mRNA. The results are representative of three individual experiments. C , Western blotting of Ei24 protein from isolated islets (300 islets/group) from WT and KO mice at 12 weeks of age. β-Actin served as the loading control. D , weight curves for WT and KO mice. The mean ± S.D. ( error bars ) of 15 mice is shown. E , concentration of the basic glucose curve for WT and KO mice. The mean ± S.D. of 10 mice is shown. F and G , glucose tolerance test ( GTT ) results of 10-week-old male ( F ) and female mice ( G ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). H and I , ITT results of 10-week-old male ( H ) and female mice ( I ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). J , in vivo GSIS detection in WT and KO mice. The results are representative of five replicates for each group. K , in vitro GSIS from isolated islets (70/group) of WT and KO mice by a fast digital perfusion system. The results are representative of three individual experiments. Data are expressed as the mean ± S.D. *, p
    β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 40061 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti β actin antibody
    a Expression levels of TSPO measured in protein extracts from rat and murine glioma cell lines (9L, C6 and GL261). Western blots were normalized using the <t>anti-β-actin</t> antibody. H E staining ( b ) and immunohistochemistry for TSPO ( c ) of coronal brain sections illustrating tumour growth and high level of TSPO expression in a 9L glioma
    Anti β Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti β actin
    In vitro Trypanosoma cruzi infection induces expression of Wnt proteins, Frizzled (Fzd) receptors, and β-catenin macrophages. Bone marrow-derived macrophages were in vitro infected with T. cruzi trypomastigotes or left uninfected (NI) and then evaluated for expression of Wnt proteins and Fzd receptors at different times post-infection (pi). (A) Expression of Wnt3a and Wnt5a mRNA (relative to <t>β-actin)</t> was determined by quantitative real-time-PCR. (B) The relative abundance of Wnt3, Wnt5a, and β-actin in the cell lysates were determined by Western blot and densitometry at 12 h pi. Representative Western blot and the ratio of Wnt3a or Wnt5a to β-actin are shown. (C) Expression of Fzd4, Fzd8, Fzd9, and Fzd6 mRNA (relative to β-actin) was determined by q-PCR. The results are expressed as the average of three independent experiments ± SEM. Abbreviation: AU, arbitrary units (* P
    Anti β Actin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8076 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti β actin
    MUC1 is widely overexpressed in NSCLC cells, correlating with STAT3 activation. (A) Protein expression of MUC1-C and total and Tyr705 phosphorylated STAT3 levels were determined in 14 human NSCLC cell lines by Western blot analyses. Equal loading and transfer were shown by repeat probing with <t>β-actin.</t> (B) mRNA levels of MUC1 in a subset of cells were evaluated with real-time RT-PCR. MUC1 mRNA expression levels in each cell line were normalized to GAPDH mRNA, with expression in A549 cells set to an arbitrary value of 1. * P
    Anti β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti β actin antibody
    Protein expression levels of PERK, IRE1α and ATF6 in ovaries of aged mice. Representative western blot images (a) and quantification of PERK, IRE1α and ATF6 protein expression levels (b). <t>β-ACTIN</t> served as an internal control. Data are expressed as the mean ± SEM, expressed by error bars.
    Anti β Actin Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2512 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam mouse anti β actin
    Gastrodin (GSTD) increased osteogenic transcription Runx2, decreased adipogenic factor peroxisome proliferator-activated receptor (PPAR)γ isoform 2 and activated nuclear factor-like 2 (NRF2) pathway protein expression levels. Representative western blot analyses for total protein was analyzed by western blotting in dexamethasone (DEX)-treated MC3T3-E1 cells with or without GSTD pretreatment at different doses. The relative protein expression levels were measured using the fold-change in each protein relative to <t>β-actin</t> from the same sample. The data are expressed as the mean ± standard deviation. ## P
    Mouse Anti β Actin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti actin antibody
    ORAIs and STIMs in human kidney and regulation under diabetic condition. a Immunostaining for ORAIs in normal and diabetic kidney tissue sections. The diabetic kidney sections showing typical mesangial expansion and accumulation of mesangial matrix material. Scale bar, 100 µm. b , c Mean ± s.e.m. for the staining intensity (arbitrary unit) in proximal tubules and distal tubules, respectively. The average of nine staining fields for each patient was calculated for proximal or distal tubule staining ( n = 6 for normal kidney, n = 8 for diabetic kidney). Also see staining for STIM1 and STIM2 (Supplementary Fig. 3 ). d Primary cultured human proximal tubular epithelial cells (PTECs) were characterized by lectin staining (red). Scale bars, 50 µm. e PTECs were cultured with normal (5.5 mM) and high (25 mM) glucose for 60 h. ORAI proteins were detected by western blotting ( n = 6). f The mRNA of ORAIs was quantified by real-time PCR. The mean data were from 2–3 independent experiments ( n = 6). g The proximal tubular epithelial cells (HK-2) were treated with or without (control) insulin (10 nM) for 48 h and the mRNA was detected by real-time PCR ( n = 6). h HK-2 cells incubated with tyrosine kinase inhibitor tyrphostin A23 (30 µM) for 48 h ( n = 6). i STIM1 and STIM2 expression after insulin (10 nM) and tyrphostin A23 (30 µM) treatment for 48 h ( n = 9). The <t>β-actin</t> was used as control for relative quantification of mRNA or protein. For PCR experiments, triplicate reactions were set for each gene. The averaged data are displayed as mean ± s.e.m. and the data in e – i are normalized to control. The data sets are compared by t test. Statistical significance is indicated by * P
    Anti Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13046 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Fyn regulates IP 3 -mediated calcium responses. (A) WEHI 7.2 T cells were transfected with Fyn siRNAs (or non-targeting control siRNAs) as described in materials and methods. Fyn levels were measured by western blotting 24 hours post-transfection. β-actin

    Journal: Autophagy

    Article Title: Glucocorticoids downregulate Fyn and inhibit IP3-mediated calcium signaling to promote autophagy in T lymphocytes

    doi: 10.4161/auto.6.7.13290

    Figure Lengend Snippet: Fyn regulates IP 3 -mediated calcium responses. (A) WEHI 7.2 T cells were transfected with Fyn siRNAs (or non-targeting control siRNAs) as described in materials and methods. Fyn levels were measured by western blotting 24 hours post-transfection. β-actin

    Article Snippet: The following antibodies were used in this study: Fyn (Santa Cruz Biotechnology, sc-16), Lck (Santa Cruz Biotechnology, sc-433), anti-mouse CD3ε (BD Biosciences, 145-2C11), IP3 R3 (BD Biosciences, 610312), β-actin (Sigma-Aldrich, A-5441), p62 (Novus Biologicals, 8878-M03), LC3 (Novus Biologicals, NB100-2220), IP3 R1 (Novus Biologicals, NB120-5908), IP3 R2 (Novus Biologicals, NB100-2466), phospho-S6 kinase (Thr389) (Cell Signaling Technology, 9205), phospho-4EBP1 (Ser65) (Cell Signaling Technology, 9451), Total S6 kinase (Cell Signaling Technology, 9202), Total 4EBP1 (Cell Signaling Technology, 9452).

    Techniques: Transfection, Western Blot

    FL BARD1 functions in DNA damage response. SKNSH and SHSY5Y cell lines were silenced for FL BARD 1 expression upon transfection with lentiviral plasmids (shBARD1#A, shBARD1#B). Unsilenced control cells were transfected with plasmid shCTR. The efficiency of short harpin silencing was verified by western blotting, using an antibody against FL BARD1 isoform. The molecular weight of FL BARD1 isoform is reported. The higher band in the blot is an aspecific staining. β-Actin levels were used as loading control (A). The detection of Υ -H2AX protein was verified in nuclear extract of silenced (shBARD1) and unsilenced control (shCTR) cells, by western blotting. Antibody against histone H3 was used as loading control (B). SKNSH shBARD1 and shCTR cells (C) and SHSY5Y shBARD1 and shCTR cells (D) were treated with 5 Gy IR. The expression of Υ- H2AX was measured by western blotting in a time-course (30 min, 1h, 3h, 6h, 12h, 24h, 36h, 48h) after IR. The integral optical density (IOD) of Υ- H2AX protein bands were measured and normalized respect to loading control protein band H3. The arrows indicate the higher increment of Υ- H2AX in each cell line. The experiments were repeated twice.

    Journal: Journal of Cancer

    Article Title: Functional characterization of full-length BARD1 strengthens its role as a tumor suppressor in neuroblastoma

    doi: 10.7150/jca.36164

    Figure Lengend Snippet: FL BARD1 functions in DNA damage response. SKNSH and SHSY5Y cell lines were silenced for FL BARD 1 expression upon transfection with lentiviral plasmids (shBARD1#A, shBARD1#B). Unsilenced control cells were transfected with plasmid shCTR. The efficiency of short harpin silencing was verified by western blotting, using an antibody against FL BARD1 isoform. The molecular weight of FL BARD1 isoform is reported. The higher band in the blot is an aspecific staining. β-Actin levels were used as loading control (A). The detection of Υ -H2AX protein was verified in nuclear extract of silenced (shBARD1) and unsilenced control (shCTR) cells, by western blotting. Antibody against histone H3 was used as loading control (B). SKNSH shBARD1 and shCTR cells (C) and SHSY5Y shBARD1 and shCTR cells (D) were treated with 5 Gy IR. The expression of Υ- H2AX was measured by western blotting in a time-course (30 min, 1h, 3h, 6h, 12h, 24h, 36h, 48h) after IR. The integral optical density (IOD) of Υ- H2AX protein bands were measured and normalized respect to loading control protein band H3. The arrows indicate the higher increment of Υ- H2AX in each cell line. The experiments were repeated twice.

    Article Snippet: Mouse monoclonal anti-β-Actin antibody (cod-A5441, Sigma-Aldrich, 1:6000) and anti-H3 (cod-06-755, Millipore, 1:1000), were used as loading control for cytosol and nuclei extracts respectively.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Molecular Weight, Staining

    FL BARD1 functions in regulating apoptosis. Phospho-p53 and p53 and β-Actin protein levels were verified by western blotting in shBARD1 and shCTR cells, in SKNSH (24 hours post-IR) and SHSY5Y (36 hours post-IR) cell lines (A). Caspase-3 activity was evaluated in SKNSH shBARD1 and shCTR IR and V cells (B) and in SHSY5Y shBARD1 and shCTR IR and V cells (C). The asterisk is indicative of p-value ≤ 0.05. The experiments were repeated twice.

    Journal: Journal of Cancer

    Article Title: Functional characterization of full-length BARD1 strengthens its role as a tumor suppressor in neuroblastoma

    doi: 10.7150/jca.36164

    Figure Lengend Snippet: FL BARD1 functions in regulating apoptosis. Phospho-p53 and p53 and β-Actin protein levels were verified by western blotting in shBARD1 and shCTR cells, in SKNSH (24 hours post-IR) and SHSY5Y (36 hours post-IR) cell lines (A). Caspase-3 activity was evaluated in SKNSH shBARD1 and shCTR IR and V cells (B) and in SHSY5Y shBARD1 and shCTR IR and V cells (C). The asterisk is indicative of p-value ≤ 0.05. The experiments were repeated twice.

    Article Snippet: Mouse monoclonal anti-β-Actin antibody (cod-A5441, Sigma-Aldrich, 1:6000) and anti-H3 (cod-06-755, Millipore, 1:1000), were used as loading control for cytosol and nuclei extracts respectively.

    Techniques: Western Blot, Activity Assay

    Knockdown of AQP5 suppressed the ability of migration and invasion in HCT116 and SW480 cells. a The ability of migration was measured by scratch wound healing assay after knockdown of AQP5, the migration rate was determined. b The invasiveness of HCT116 and SW480 cells with and without AQP5 silencing was evaluated by transwell assay, invasive cells were counted and expressed as fold change compared to the Mock group. c The expression levels of MMP-2 and MMP-9 were detected by immunoblotting after knockdown of AQP5, the bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. d The activities of MMP-2 and MMP-9 in AQP5-silenced and control cells were assessed by gelatin zymography. Data were presented as the mean ± SD. * p

    Journal: Cytotechnology

    Article Title: Anti-cancer effect of Aquaporin 5 silencing in colorectal cancer cells in association with inhibition of Wnt/β-catenin pathway

    doi: 10.1007/s10616-017-0147-7

    Figure Lengend Snippet: Knockdown of AQP5 suppressed the ability of migration and invasion in HCT116 and SW480 cells. a The ability of migration was measured by scratch wound healing assay after knockdown of AQP5, the migration rate was determined. b The invasiveness of HCT116 and SW480 cells with and without AQP5 silencing was evaluated by transwell assay, invasive cells were counted and expressed as fold change compared to the Mock group. c The expression levels of MMP-2 and MMP-9 were detected by immunoblotting after knockdown of AQP5, the bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. d The activities of MMP-2 and MMP-9 in AQP5-silenced and control cells were assessed by gelatin zymography. Data were presented as the mean ± SD. * p

    Article Snippet: Proteins from the cell lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto Polyvinylidene Fluoride (PVDF) membranes, and immunoblotted respectively with antibodies against AQP5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-514022), β-catenin (BOSTER Biological Technology, Pleasanton, CA, USA, BA0426), MMP2 (BOSTER, BA0569), MMP9 (BOSTER, BA0573), E-cadherin (BOSTER, BA0474), Vimentin (Bioss Antibodies, Woburn, MA, USA, bs-8533R), N-cadherin (BOSTER, BA0673), uPA (Bioss, bs-1927R), TIMP-1 (Bioss, bs-0415R), TIMP-2 (Bioss, bs-10395R), Snail (Bioss, bs-1371R), Wnt1 (BOSTER, BA3158-2), β-actin (Santa Cruz Biotechnology, sc-47778).

    Techniques: Migration, Wound Healing Assay, Transwell Assay, Expressing, Zymography

    Silencing of AQP5 inhibited Wnt/β-catenin signal transduction. a The expression levels of Wnt1 and β-catenin were detected by western blotting after silencing AQP5 in HCT116 and SW480 cells. b Following transfection of β-catenin S33Y in AQP5-silenced HCT116 and SW480 cells, the expression of β-catenin, E-cadherin, Vimentin, N-cadherin and Snail was determined by western blotting. The bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. Data were presented as the mean ± SD. * p

    Journal: Cytotechnology

    Article Title: Anti-cancer effect of Aquaporin 5 silencing in colorectal cancer cells in association with inhibition of Wnt/β-catenin pathway

    doi: 10.1007/s10616-017-0147-7

    Figure Lengend Snippet: Silencing of AQP5 inhibited Wnt/β-catenin signal transduction. a The expression levels of Wnt1 and β-catenin were detected by western blotting after silencing AQP5 in HCT116 and SW480 cells. b Following transfection of β-catenin S33Y in AQP5-silenced HCT116 and SW480 cells, the expression of β-catenin, E-cadherin, Vimentin, N-cadherin and Snail was determined by western blotting. The bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. Data were presented as the mean ± SD. * p

    Article Snippet: Proteins from the cell lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto Polyvinylidene Fluoride (PVDF) membranes, and immunoblotted respectively with antibodies against AQP5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-514022), β-catenin (BOSTER Biological Technology, Pleasanton, CA, USA, BA0426), MMP2 (BOSTER, BA0569), MMP9 (BOSTER, BA0573), E-cadherin (BOSTER, BA0474), Vimentin (Bioss Antibodies, Woburn, MA, USA, bs-8533R), N-cadherin (BOSTER, BA0673), uPA (Bioss, bs-1927R), TIMP-1 (Bioss, bs-0415R), TIMP-2 (Bioss, bs-10395R), Snail (Bioss, bs-1371R), Wnt1 (BOSTER, BA3158-2), β-actin (Santa Cruz Biotechnology, sc-47778).

    Techniques: Transduction, Expressing, Western Blot, Transfection

    Knockdown of AQP5 regulated the expression of EMT-related proteins in HCT116 and SW480 cells. a The expression levels of E-cadherin, Vimentin, N-cadherin, uPA, TIMP-1, TIMP-2 and Snail were measured by western-blot analysis after knockdown of AQP5, the bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. b Immunofluorescence staining was performed to detect the expression of E-cadherin and Snail in HCT116 and SW480 cells with and without AQP5 silencing. Data were presented as the mean ± SD. * p

    Journal: Cytotechnology

    Article Title: Anti-cancer effect of Aquaporin 5 silencing in colorectal cancer cells in association with inhibition of Wnt/β-catenin pathway

    doi: 10.1007/s10616-017-0147-7

    Figure Lengend Snippet: Knockdown of AQP5 regulated the expression of EMT-related proteins in HCT116 and SW480 cells. a The expression levels of E-cadherin, Vimentin, N-cadherin, uPA, TIMP-1, TIMP-2 and Snail were measured by western-blot analysis after knockdown of AQP5, the bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. b Immunofluorescence staining was performed to detect the expression of E-cadherin and Snail in HCT116 and SW480 cells with and without AQP5 silencing. Data were presented as the mean ± SD. * p

    Article Snippet: Proteins from the cell lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto Polyvinylidene Fluoride (PVDF) membranes, and immunoblotted respectively with antibodies against AQP5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-514022), β-catenin (BOSTER Biological Technology, Pleasanton, CA, USA, BA0426), MMP2 (BOSTER, BA0569), MMP9 (BOSTER, BA0573), E-cadherin (BOSTER, BA0474), Vimentin (Bioss Antibodies, Woburn, MA, USA, bs-8533R), N-cadherin (BOSTER, BA0673), uPA (Bioss, bs-1927R), TIMP-1 (Bioss, bs-0415R), TIMP-2 (Bioss, bs-10395R), Snail (Bioss, bs-1371R), Wnt1 (BOSTER, BA3158-2), β-actin (Santa Cruz Biotechnology, sc-47778).

    Techniques: Expressing, Western Blot, Immunofluorescence, Staining

    Knockdown of AQP5 in HCT116 and SW480 cells. a The expression of AQP5 protein was assessed by western-blot assay after knockdown of AQP5, the bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. b The expression of AQP5 mRNA was measured by real-time PCR after silencing AQP5. Data were presented as the mean ± SD. ** p

    Journal: Cytotechnology

    Article Title: Anti-cancer effect of Aquaporin 5 silencing in colorectal cancer cells in association with inhibition of Wnt/β-catenin pathway

    doi: 10.1007/s10616-017-0147-7

    Figure Lengend Snippet: Knockdown of AQP5 in HCT116 and SW480 cells. a The expression of AQP5 protein was assessed by western-blot assay after knockdown of AQP5, the bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. b The expression of AQP5 mRNA was measured by real-time PCR after silencing AQP5. Data were presented as the mean ± SD. ** p

    Article Snippet: Proteins from the cell lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto Polyvinylidene Fluoride (PVDF) membranes, and immunoblotted respectively with antibodies against AQP5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-514022), β-catenin (BOSTER Biological Technology, Pleasanton, CA, USA, BA0426), MMP2 (BOSTER, BA0569), MMP9 (BOSTER, BA0573), E-cadherin (BOSTER, BA0474), Vimentin (Bioss Antibodies, Woburn, MA, USA, bs-8533R), N-cadherin (BOSTER, BA0673), uPA (Bioss, bs-1927R), TIMP-1 (Bioss, bs-0415R), TIMP-2 (Bioss, bs-10395R), Snail (Bioss, bs-1371R), Wnt1 (BOSTER, BA3158-2), β-actin (Santa Cruz Biotechnology, sc-47778).

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

    The combination of berberine with resveratrol increased LDLR expression in HepG2 cells. Cells were cultured in 6-well plate for 24 h with a 3 × 10 5 cell density, and then culture medium were replaced by fresh medium containing different concentration of FBS indicated in A, or 1% FBS and drugs indicated in B for another 24 h followed by extraction of the total proteins from the cells. A : the effect of different concentration FBS on LDLR expression were analyzed by western blot assay. Then the band intensity was quantified by grey scanning analysis, and the intensity ratio of LDLR to β-actin in 0% FBS group was set to 1. *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Combination of Berberine with Resveratrol Improves the Lipid-Lowering Efficacy

    doi: 10.3390/ijms19123903

    Figure Lengend Snippet: The combination of berberine with resveratrol increased LDLR expression in HepG2 cells. Cells were cultured in 6-well plate for 24 h with a 3 × 10 5 cell density, and then culture medium were replaced by fresh medium containing different concentration of FBS indicated in A, or 1% FBS and drugs indicated in B for another 24 h followed by extraction of the total proteins from the cells. A : the effect of different concentration FBS on LDLR expression were analyzed by western blot assay. Then the band intensity was quantified by grey scanning analysis, and the intensity ratio of LDLR to β-actin in 0% FBS group was set to 1. *** p

    Article Snippet: Goat antibodies directed against LDLR (C-7, sc-18823), HRP-labeled anti-goat IgG (sc-2354) and anti-β-actin antibody (sc-8432) were from Santa Cruz Biotechnology (Heidelberg, German).

    Techniques: Expressing, Cell Culture, Concentration Assay, Western Blot

    Single cell traction force measurement in multiple cell types using the protein micropillar arrays. ( A–I ) Fluorescence/Immunofluorescence staining of ( A–C ) bovine nucleus pulposus cells (bNPCs), ( D–F ) human mesenchymal stem cells (hMSCs), ( G–I ) human dermal fibroblasts (hDFs) and ( J–L ) rabbit chondrocyte, which were cultured on the protein micropillar arrays. Green: Immunofluorescence staining of beta-actin; Blue: DAPI showing the nuclei; Red: Rose Bengal showing the cross-sections of the protein micropillars. The micropillars all have a diameter of 1 μm and a reduced elastic modulus of 30 kPa, but are of different heights (i.e., 6 μm, 8 μm and 10 μm), which are equivalent to a stiffness of ( A,D,G,J ) 20.44 nN/μm, ( B,E,H,K ) 8.62 nN/μm, and ( C,F,I,L ) 4.42 nN/μm, respectively. Scale bars, 10 μm. ( M ) Bar chart showing the single cell total traction force of different types of cells cultured on protein micropillar arrays of different stiffness: bovine nucleus pulposus cells (bNPCs, P2 ~ P4); human mesenchymal stem cells (hMSCs, P3 ~ P5); human dermal fibroblasts (hDF, P5 ~ P15) and rabbit chondrocytes (rChondrocytes, P2 ~ P4) cultured for 3 days on protein micropillars (n = 10).

    Journal: Scientific Reports

    Article Title: Multiphoton photochemical crosslinking-based fabrication of protein micropatterns with controllable mechanical properties for single cell traction force measurements

    doi: 10.1038/srep20063

    Figure Lengend Snippet: Single cell traction force measurement in multiple cell types using the protein micropillar arrays. ( A–I ) Fluorescence/Immunofluorescence staining of ( A–C ) bovine nucleus pulposus cells (bNPCs), ( D–F ) human mesenchymal stem cells (hMSCs), ( G–I ) human dermal fibroblasts (hDFs) and ( J–L ) rabbit chondrocyte, which were cultured on the protein micropillar arrays. Green: Immunofluorescence staining of beta-actin; Blue: DAPI showing the nuclei; Red: Rose Bengal showing the cross-sections of the protein micropillars. The micropillars all have a diameter of 1 μm and a reduced elastic modulus of 30 kPa, but are of different heights (i.e., 6 μm, 8 μm and 10 μm), which are equivalent to a stiffness of ( A,D,G,J ) 20.44 nN/μm, ( B,E,H,K ) 8.62 nN/μm, and ( C,F,I,L ) 4.42 nN/μm, respectively. Scale bars, 10 μm. ( M ) Bar chart showing the single cell total traction force of different types of cells cultured on protein micropillar arrays of different stiffness: bovine nucleus pulposus cells (bNPCs, P2 ~ P4); human mesenchymal stem cells (hMSCs, P3 ~ P5); human dermal fibroblasts (hDF, P5 ~ P15) and rabbit chondrocytes (rChondrocytes, P2 ~ P4) cultured for 3 days on protein micropillars (n = 10).

    Article Snippet: Samples were blocked in 5% BSA in PBS for 30 min and then incubated with a primary monoclonal anti-β-actin antibody (1:100, A2228, Sigma-Aldrich) at 4 °C overnight.

    Techniques: Fluorescence, Immunofluorescence, Staining, Cell Culture

    Loss of Ei24 impairs glucose homeostasis. A , expression of Ei24 in islets from ob/ob mice, GK rats at 4 months of age, and C57BL/6 mice fed an HFD for 2 weeks and 8 weeks. β-Actin served as the loading control. CD , chow diet. B , total RNA was prepared from islets of WT and KO mice at 8 weeks of age. The transcription levels of Ei24 mRNA are normalized to β-actin mRNA. The results are representative of three individual experiments. C , Western blotting of Ei24 protein from isolated islets (300 islets/group) from WT and KO mice at 12 weeks of age. β-Actin served as the loading control. D , weight curves for WT and KO mice. The mean ± S.D. ( error bars ) of 15 mice is shown. E , concentration of the basic glucose curve for WT and KO mice. The mean ± S.D. of 10 mice is shown. F and G , glucose tolerance test ( GTT ) results of 10-week-old male ( F ) and female mice ( G ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). H and I , ITT results of 10-week-old male ( H ) and female mice ( I ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). J , in vivo GSIS detection in WT and KO mice. The results are representative of five replicates for each group. K , in vitro GSIS from isolated islets (70/group) of WT and KO mice by a fast digital perfusion system. The results are representative of three individual experiments. Data are expressed as the mean ± S.D. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Etoposide-induced protein 2.4 functions as a regulator of the calcium ATPase and protects pancreatic β-cell survival

    doi: 10.1074/jbc.RA118.002399

    Figure Lengend Snippet: Loss of Ei24 impairs glucose homeostasis. A , expression of Ei24 in islets from ob/ob mice, GK rats at 4 months of age, and C57BL/6 mice fed an HFD for 2 weeks and 8 weeks. β-Actin served as the loading control. CD , chow diet. B , total RNA was prepared from islets of WT and KO mice at 8 weeks of age. The transcription levels of Ei24 mRNA are normalized to β-actin mRNA. The results are representative of three individual experiments. C , Western blotting of Ei24 protein from isolated islets (300 islets/group) from WT and KO mice at 12 weeks of age. β-Actin served as the loading control. D , weight curves for WT and KO mice. The mean ± S.D. ( error bars ) of 15 mice is shown. E , concentration of the basic glucose curve for WT and KO mice. The mean ± S.D. of 10 mice is shown. F and G , glucose tolerance test ( GTT ) results of 10-week-old male ( F ) and female mice ( G ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). H and I , ITT results of 10-week-old male ( H ) and female mice ( I ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). J , in vivo GSIS detection in WT and KO mice. The results are representative of five replicates for each group. K , in vitro GSIS from isolated islets (70/group) of WT and KO mice by a fast digital perfusion system. The results are representative of three individual experiments. Data are expressed as the mean ± S.D. *, p

    Article Snippet: Antibodies against Ei24 (Sigma), proinsulin (Novus Biologicals), insulin (Abcam), c-PARP, total PARP (t-PARP), c-caspase-3, total caspase 3 (t-caspase-3), phosphorylated AMPK (p-AMPK), total AMPK (t-AMPK), p-ACC, total ACC (t-ACC), phosphorylated CAMKK2 (p-CAMKK2), ATP2a2, and β-actin (Cell Signaling Technology) were used, according to the manufacturer's protocols.

    Techniques: Expressing, Mouse Assay, Western Blot, Isolation, Concentration Assay, In Vivo, In Vitro

    Loss of Ei24 impairs AMPK activation and induces apoptotic cell death. A , ATP content of islets (40 islets/group), which were cultured in RPMI medium 1640 with 10% FBS from WT and KO mice at 8 weeks of age. Data were normalized to protein content. Data were obtained from three independent experiments. B , Western blotting for p-AMPK, t-AMPK, p-ACC, and t-ACC in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. C , Western blotting for p-CAMKK2 in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. D , Western blotting for p-AMPK, t-AMPK, c-PARP, t-PARP, c-caspase-3, and t-caspase-3 in the isolated islets (150 islets/group) with or without AICAR treatment from WT and KO mice at 8–10 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. Data are expressed as the mean ± S.D. ( error bars ): WT versus KO (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: Etoposide-induced protein 2.4 functions as a regulator of the calcium ATPase and protects pancreatic β-cell survival

    doi: 10.1074/jbc.RA118.002399

    Figure Lengend Snippet: Loss of Ei24 impairs AMPK activation and induces apoptotic cell death. A , ATP content of islets (40 islets/group), which were cultured in RPMI medium 1640 with 10% FBS from WT and KO mice at 8 weeks of age. Data were normalized to protein content. Data were obtained from three independent experiments. B , Western blotting for p-AMPK, t-AMPK, p-ACC, and t-ACC in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. C , Western blotting for p-CAMKK2 in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. D , Western blotting for p-AMPK, t-AMPK, c-PARP, t-PARP, c-caspase-3, and t-caspase-3 in the isolated islets (150 islets/group) with or without AICAR treatment from WT and KO mice at 8–10 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. Data are expressed as the mean ± S.D. ( error bars ): WT versus KO (*, p

    Article Snippet: Antibodies against Ei24 (Sigma), proinsulin (Novus Biologicals), insulin (Abcam), c-PARP, total PARP (t-PARP), c-caspase-3, total caspase 3 (t-caspase-3), phosphorylated AMPK (p-AMPK), total AMPK (t-AMPK), p-ACC, total ACC (t-ACC), phosphorylated CAMKK2 (p-CAMKK2), ATP2a2, and β-actin (Cell Signaling Technology) were used, according to the manufacturer's protocols.

    Techniques: Activation Assay, Cell Culture, Mouse Assay, Western Blot, Isolation

    Loss of Ei24 causes apoptosis of pancreatic β cells. A , morphologies of islets. The islets from KO mice became uncompact and transparent compared with the WT islets. B , representative images of H E staining of islets from WT and KO mice at the age of 12 weeks. The degenerative β cells are indicated by black arrowheads. C , representative sections of islets stained for insulin ( red ) and nuclei ( blue ) from WT and KO mice at 12 weeks of age. Scale bars , 10 μm. D , density of β cells determined by counting the number of β cells in islets ( n = 40/group) from WT and KO mice. E , EM micrographs of WT and KO pancreatic β cells. The bottom panel in E shows enlargement of the boxed area in the top panel . The arrows indicate the dense core vesicles. F , the number of dense core vesicles ( DCV ) in pancreatic β cells was counted using Imaris software (cell numbers, n = 48/group). G , Western blotting for proinsulin in the isolated islets (30 islets/group) from WT and KO mice at 8 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. H , Western blotting for insulin in the isolated islets (60 islets/group) from WT and KO mice at 8 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. I , Western blotting for c-PARP, t-PARP, c-caspase-3, and t-caspase-3 in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. β-Actin served as the loading control. Data were obtained from five independent experiments. Data are expressed as the mean ± S.D. ( error bars ). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Etoposide-induced protein 2.4 functions as a regulator of the calcium ATPase and protects pancreatic β-cell survival

    doi: 10.1074/jbc.RA118.002399

    Figure Lengend Snippet: Loss of Ei24 causes apoptosis of pancreatic β cells. A , morphologies of islets. The islets from KO mice became uncompact and transparent compared with the WT islets. B , representative images of H E staining of islets from WT and KO mice at the age of 12 weeks. The degenerative β cells are indicated by black arrowheads. C , representative sections of islets stained for insulin ( red ) and nuclei ( blue ) from WT and KO mice at 12 weeks of age. Scale bars , 10 μm. D , density of β cells determined by counting the number of β cells in islets ( n = 40/group) from WT and KO mice. E , EM micrographs of WT and KO pancreatic β cells. The bottom panel in E shows enlargement of the boxed area in the top panel . The arrows indicate the dense core vesicles. F , the number of dense core vesicles ( DCV ) in pancreatic β cells was counted using Imaris software (cell numbers, n = 48/group). G , Western blotting for proinsulin in the isolated islets (30 islets/group) from WT and KO mice at 8 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. H , Western blotting for insulin in the isolated islets (60 islets/group) from WT and KO mice at 8 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. I , Western blotting for c-PARP, t-PARP, c-caspase-3, and t-caspase-3 in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. β-Actin served as the loading control. Data were obtained from five independent experiments. Data are expressed as the mean ± S.D. ( error bars ). *, p

    Article Snippet: Antibodies against Ei24 (Sigma), proinsulin (Novus Biologicals), insulin (Abcam), c-PARP, total PARP (t-PARP), c-caspase-3, total caspase 3 (t-caspase-3), phosphorylated AMPK (p-AMPK), total AMPK (t-AMPK), p-ACC, total ACC (t-ACC), phosphorylated CAMKK2 (p-CAMKK2), ATP2a2, and β-actin (Cell Signaling Technology) were used, according to the manufacturer's protocols.

    Techniques: Mouse Assay, Staining, Software, Western Blot, Isolation

    KPNB1 expression is associated with human prostate cancer progression. A) The expression level of KPNB1 in human prostate cancer samples (N=498) and normal prostate tissues (N=52). Samples are from TCGA dataset. B) The expression level of KPNB1 human prostate cancer samples with different Gleason scores. Samples are from TCGA dataset. C) The expression level of KPNB1 human prostate cancer samples with different pathological stages. Samples are from TCGA dataset. D) The expression level of KPNB1 of localized and metastasis human prostate cancer samples. Samples are from GEO dataset. E) qPCR results showing relative expression level of KPNB1 RNA in indicated cell lines. FTH1 was used as internal control. F) Immunoblotting results showing protein level of KPNB1 in indicated cell lines. β-actin was used as internal control. G) Representative images of IHC staining of KPNB1 on human prostate cancer TMA slides. H) Statistical results of KPNB1 positive cells on the TMA slides. * p

    Journal: Oncogene

    Article Title: Inhibition of Karyopherin beta 1 suppresses prostate cancer growth

    doi: 10.1038/s41388-019-0745-2

    Figure Lengend Snippet: KPNB1 expression is associated with human prostate cancer progression. A) The expression level of KPNB1 in human prostate cancer samples (N=498) and normal prostate tissues (N=52). Samples are from TCGA dataset. B) The expression level of KPNB1 human prostate cancer samples with different Gleason scores. Samples are from TCGA dataset. C) The expression level of KPNB1 human prostate cancer samples with different pathological stages. Samples are from TCGA dataset. D) The expression level of KPNB1 of localized and metastasis human prostate cancer samples. Samples are from GEO dataset. E) qPCR results showing relative expression level of KPNB1 RNA in indicated cell lines. FTH1 was used as internal control. F) Immunoblotting results showing protein level of KPNB1 in indicated cell lines. β-actin was used as internal control. G) Representative images of IHC staining of KPNB1 on human prostate cancer TMA slides. H) Statistical results of KPNB1 positive cells on the TMA slides. * p

    Article Snippet: Anti-CDK1, anti-RCC1, anti-pRCC1, anti-β-actin, anti-Ki67, and anti-caspase 3 primary antibodies and all secondary antibodies were purchased from Cell signaling technology (Danvers, MA, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining

    Inhibition of KPNB1 using importazole suppresses PCa growth. A and B) Crystal violet staining showing the growth of indicated cell lines that were treated with 10 μM or 20 μM of importazole for 24 hours, 48 hours or 72 hours. DMSO was used as vehicle control. C) MTT assay showing relative cell viabilities of PC3 or C42B that was treated with importazole of indicated concentrations for 48 hours. DMSO was used as vehicle control. D and E) Flow cytometry showing the cell cycle stage distribution of indicated cell lines that were treated with 10 μM or 20 μM of importazole for 48 hours. DMSO was used as vehicle control. F) Immunoblotting results showing protein level of Cyclin B1, CDK1, pRCC1 and Cyclin D1 of indicated cell lines that were treated with DMSO, 10 μM or 20 μM of importazole for 48 hours. β-actin was used as internal control. G) Immunoblotting results showing nuclear distribution of c-Myc of indicated cell lines that were treated with 10 μM or 20 μM of importazole for 48 hours. DMSO was used as vehicle control. Lamin A/C was used as loading control of nuclear protein. * p

    Journal: Oncogene

    Article Title: Inhibition of Karyopherin beta 1 suppresses prostate cancer growth

    doi: 10.1038/s41388-019-0745-2

    Figure Lengend Snippet: Inhibition of KPNB1 using importazole suppresses PCa growth. A and B) Crystal violet staining showing the growth of indicated cell lines that were treated with 10 μM or 20 μM of importazole for 24 hours, 48 hours or 72 hours. DMSO was used as vehicle control. C) MTT assay showing relative cell viabilities of PC3 or C42B that was treated with importazole of indicated concentrations for 48 hours. DMSO was used as vehicle control. D and E) Flow cytometry showing the cell cycle stage distribution of indicated cell lines that were treated with 10 μM or 20 μM of importazole for 48 hours. DMSO was used as vehicle control. F) Immunoblotting results showing protein level of Cyclin B1, CDK1, pRCC1 and Cyclin D1 of indicated cell lines that were treated with DMSO, 10 μM or 20 μM of importazole for 48 hours. β-actin was used as internal control. G) Immunoblotting results showing nuclear distribution of c-Myc of indicated cell lines that were treated with 10 μM or 20 μM of importazole for 48 hours. DMSO was used as vehicle control. Lamin A/C was used as loading control of nuclear protein. * p

    Article Snippet: Anti-CDK1, anti-RCC1, anti-pRCC1, anti-β-actin, anti-Ki67, and anti-caspase 3 primary antibodies and all secondary antibodies were purchased from Cell signaling technology (Danvers, MA, USA).

    Techniques: Inhibition, Staining, MTT Assay, Flow Cytometry, Cytometry

    The effect of BV and melittin on the expression of apoptosis regulatory proteins in A375SM melanoma cells. Cells were treated with BV and melittin for 24 h, and the expression levels of cleaved caspase-3 and cleaved caspase-9 were detected by Western blotting. The levels of β-actin were used as an internal control. Each value represents the mean ± SE from three independent experiments.

    Journal: Molecules

    Article Title: Bee Venom and Its Peptide Component Melittin Suppress Growth and Migration of Melanoma Cells via Inhibition of PI3K/AKT/mTOR and MAPK Pathways

    doi: 10.3390/molecules24050929

    Figure Lengend Snippet: The effect of BV and melittin on the expression of apoptosis regulatory proteins in A375SM melanoma cells. Cells were treated with BV and melittin for 24 h, and the expression levels of cleaved caspase-3 and cleaved caspase-9 were detected by Western blotting. The levels of β-actin were used as an internal control. Each value represents the mean ± SE from three independent experiments.

    Article Snippet: Anti-phospho-PI3K, anti-PI3K, anti-phospho-AKT, anti-AKT, anti-phospho-mTOR, anti-mTOR, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-p38, anti-p38, anti-cleaved caspase-3, anti-cleaved capase-9, anti-MITF, anti-MMP-2, anti-MMP-9 and anti-β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Expressing, Western Blot

    The effect of BV and melittin on the regulation of PI3K/AKT/mTOR and MAPK pathways. A375SM melanoma cells were treated with ( A ) BV, melittin and ( B ) MG132, and the protein levels were detected by Western blot analysis using specific antibodies. The levels of β-actin were used as an internal control. Each value represents the mean ± SE from three independent experiments.

    Journal: Molecules

    Article Title: Bee Venom and Its Peptide Component Melittin Suppress Growth and Migration of Melanoma Cells via Inhibition of PI3K/AKT/mTOR and MAPK Pathways

    doi: 10.3390/molecules24050929

    Figure Lengend Snippet: The effect of BV and melittin on the regulation of PI3K/AKT/mTOR and MAPK pathways. A375SM melanoma cells were treated with ( A ) BV, melittin and ( B ) MG132, and the protein levels were detected by Western blot analysis using specific antibodies. The levels of β-actin were used as an internal control. Each value represents the mean ± SE from three independent experiments.

    Article Snippet: Anti-phospho-PI3K, anti-PI3K, anti-phospho-AKT, anti-AKT, anti-phospho-mTOR, anti-mTOR, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-p38, anti-p38, anti-cleaved caspase-3, anti-cleaved capase-9, anti-MITF, anti-MMP-2, anti-MMP-9 and anti-β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Western Blot

    Effects of PZH on HIF-1 α , VEGF-A, and VEGFR2 expression in HUVECs. (a) mRNA expression levels of HIF-1 α were analyzed by RT-PCR, and α -tubulin was used as a loading control. (b) Protein expression levels of HIF-1 α and VEGFR2 were analyzed by Western blot, and β -actin was used as a loading control. (c) Secreted VEGF protein was analyzed by ELISA. # A significant difference compared with normoxic control ( P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Pien Tze Huang Inhibits Hypoxia-Induced Angiogenesis via HIF-1α/VEGF-A Pathway in Colorectal Cancer

    doi: 10.1155/2015/454279

    Figure Lengend Snippet: Effects of PZH on HIF-1 α , VEGF-A, and VEGFR2 expression in HUVECs. (a) mRNA expression levels of HIF-1 α were analyzed by RT-PCR, and α -tubulin was used as a loading control. (b) Protein expression levels of HIF-1 α and VEGFR2 were analyzed by Western blot, and β -actin was used as a loading control. (c) Secreted VEGF protein was analyzed by ELISA. # A significant difference compared with normoxic control ( P

    Article Snippet: HIF-1α , β -actin antibodies, and horseradish peroxidase- (HRP-) conjugated secondary antibodies were purchased from Cell Signaling (Beverly, MA, USA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

    Effects of PZH on the expression of HIF-1 α and VEGF-A in HCT-8. (a) mRNA expression levels of HIF-1 α were analyzed by RT-PCR, and α -tubulin was used as a loading control. (b) Protein expression levels of HIF-1 α were analyzed by Western blot, and β -actin was used as a loading control. (c) Secreted VEGF protein was analyzed by ELISA. # A significant difference compared with normoxic control ( P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Pien Tze Huang Inhibits Hypoxia-Induced Angiogenesis via HIF-1α/VEGF-A Pathway in Colorectal Cancer

    doi: 10.1155/2015/454279

    Figure Lengend Snippet: Effects of PZH on the expression of HIF-1 α and VEGF-A in HCT-8. (a) mRNA expression levels of HIF-1 α were analyzed by RT-PCR, and α -tubulin was used as a loading control. (b) Protein expression levels of HIF-1 α were analyzed by Western blot, and β -actin was used as a loading control. (c) Secreted VEGF protein was analyzed by ELISA. # A significant difference compared with normoxic control ( P

    Article Snippet: HIF-1α , β -actin antibodies, and horseradish peroxidase- (HRP-) conjugated secondary antibodies were purchased from Cell Signaling (Beverly, MA, USA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

    Regulation of ALDH1A1 expression by tamoxifen-activated ERα36. (A) Putative sites in ERα36 involved in interaction with 4-OHT. All aa residues close to 4-OHT in less than 4 Å are shown by lines. The analysis was performed with Discovery Studio 2.0 (Accelrys Software Inc.). (B) Binding of 4-OHT to purified GST-ERα36 fusion protein. GST-ERα36 was immobilized to an SPR sensor chip by GST capturing and 4-OHT was introduced as the soluble-phase analyte. The sensorgrams reached equilibrium and rapidly returned to baseline, demonstrating quick interaction kinetics between GST-ERα36 and 4-OHT. The KD was estimated as 11.6 ± 1.0 μM using the Biacore Evaluation Software. (C) Nuclear localization of ERα36 (green) in MDA-MB 436 cells after treatment with 4-OHT (1 μM) for 20 and 40 min. Heochst (blue) was used for nuclear staining. Scale bar, 20 μm. (D) Western blot of ERα36 in the cytoplasm or nuclei of MDA-MB 436-ALDH1 high cells after 4-OHT or E2 treatment. Lamin-B1 was used as a nuclear protein control, β-actin as a cytoplasm protein control. C, cytoplasm; N, nuclei. (E) HS578 ERE-luciferase assays showing the transcriptional ability of ERα36 activated by E2 or 4-OHT. ERα36 or ERα66 was transfected into HS578 cells along with an ERE-luciferase element. The transcriptional activity was measured. Error bars represent SEM from mean of triplicate samples. (F) Two potential ERE-binding sites in the ALDH1A1 promoter as analyzed by Transcription Element Search System. (G) ChIP/PCR analysis of MDA-MB-436 cell lysates showing endogenous ERα36 bound to ALDH1A1 promoter after treatment with E2 (1 nM) or 4-OHT (1 μM). An unrelated mouse IgG was used as an immunoprecipitation control. * P

    Journal: Cell Research

    Article Title: Tamoxifen enhances stemness and promotes metastasis of ERα36+ breast cancer by upregulating ALDH1A1 in cancer cells

    doi: 10.1038/cr.2018.15

    Figure Lengend Snippet: Regulation of ALDH1A1 expression by tamoxifen-activated ERα36. (A) Putative sites in ERα36 involved in interaction with 4-OHT. All aa residues close to 4-OHT in less than 4 Å are shown by lines. The analysis was performed with Discovery Studio 2.0 (Accelrys Software Inc.). (B) Binding of 4-OHT to purified GST-ERα36 fusion protein. GST-ERα36 was immobilized to an SPR sensor chip by GST capturing and 4-OHT was introduced as the soluble-phase analyte. The sensorgrams reached equilibrium and rapidly returned to baseline, demonstrating quick interaction kinetics between GST-ERα36 and 4-OHT. The KD was estimated as 11.6 ± 1.0 μM using the Biacore Evaluation Software. (C) Nuclear localization of ERα36 (green) in MDA-MB 436 cells after treatment with 4-OHT (1 μM) for 20 and 40 min. Heochst (blue) was used for nuclear staining. Scale bar, 20 μm. (D) Western blot of ERα36 in the cytoplasm or nuclei of MDA-MB 436-ALDH1 high cells after 4-OHT or E2 treatment. Lamin-B1 was used as a nuclear protein control, β-actin as a cytoplasm protein control. C, cytoplasm; N, nuclei. (E) HS578 ERE-luciferase assays showing the transcriptional ability of ERα36 activated by E2 or 4-OHT. ERα36 or ERα66 was transfected into HS578 cells along with an ERE-luciferase element. The transcriptional activity was measured. Error bars represent SEM from mean of triplicate samples. (F) Two potential ERE-binding sites in the ALDH1A1 promoter as analyzed by Transcription Element Search System. (G) ChIP/PCR analysis of MDA-MB-436 cell lysates showing endogenous ERα36 bound to ALDH1A1 promoter after treatment with E2 (1 nM) or 4-OHT (1 μM). An unrelated mouse IgG was used as an immunoprecipitation control. * P

    Article Snippet: Primary antibodies (ERα36, ERα66 and β-actin) were added overnight at 4 °C, followed by incubation with a secondary antibody at room temperature for 2 h. The antibody against β-actin as a control was from Cell Signaling Technology (#9559).

    Techniques: Expressing, Software, Binding Assay, Purification, SPR Assay, Chromatin Immunoprecipitation, Multiple Displacement Amplification, Staining, Western Blot, Luciferase, Transfection, Activity Assay, Polymerase Chain Reaction, Immunoprecipitation

    a Expression levels of TSPO measured in protein extracts from rat and murine glioma cell lines (9L, C6 and GL261). Western blots were normalized using the anti-β-actin antibody. H E staining ( b ) and immunohistochemistry for TSPO ( c ) of coronal brain sections illustrating tumour growth and high level of TSPO expression in a 9L glioma

    Journal: European Journal of Nuclear Medicine and Molecular Imaging

    Article Title: The translocator protein ligand [18F]DPA-714 images glioma and activated microglia in vivo

    doi: 10.1007/s00259-011-2041-4

    Figure Lengend Snippet: a Expression levels of TSPO measured in protein extracts from rat and murine glioma cell lines (9L, C6 and GL261). Western blots were normalized using the anti-β-actin antibody. H E staining ( b ) and immunohistochemistry for TSPO ( c ) of coronal brain sections illustrating tumour growth and high level of TSPO expression in a 9L glioma

    Article Snippet: Incubation with the primary antibodies diluted in PBST with 3% of BSA for the anti-TSPO antibody and 3% non-fat dry milk for the anti-β-actin antibody, respectively (1/10,000 dilution of the anti-rat TSPO antibody NP155 [ ] generously provided by Dr. M. Higuchi, NIRS, Japan, and 1/5,000 dilution of the anti-β-actin antibody purchased from Sigma Aldrich), was performed overnight at 4°C.

    Techniques: Expressing, Western Blot, Staining, Immunohistochemistry

    Activation of Wnt signal induces midbrain characteristics in human ESC-derived NPCs. a Efficient induction of neural rosette cells from human ESCs by co-treatment with dorsomorphin and SB431542. b Strong immunoreactivity for SOX1 and Nestin in neural rosette cells. Morphology of neural rosette cells expanding in either the absence ( c ) or the presence of 1 μM BIO ( d ). e NPCs treated with BIO maintained immunoreactivity for SOX1 and Nestin. f Treatment with BIO upregulated EN1 expression and downregulated expressions of BF1 and GBX2 in dose-dependent manner. g BIO treatment significantly increased the number of EN1-positive neural cells. h The inductive effect of BIO treatment on midbrain fate appeared to be more specific than that of FGF8. i Expression pattern of another set of regional markers ( SIX3 for forebrain; PAX2 for midbrain; and HOXA2 for hindbrain) supported the midbrain-biased fate of NPCs treated with BIO. Treatment with other known GSK3 inhibitors, 1-AKP ( j ) and LiCl ( k ), resulted in regionalization comparable to BIO treatment. l Treating NPCs with Wnt antagonists (100 ng/ml DKK-1 and 500 ng/ml frizzled-5) downregulated the endogenous level of EN1 transcript in NPCs. m Immunoblot for β-catenin and EN1 protein after introduction of two different β-catenin-specific shRNAs (shRNA-1 and shRNA-2). EN1 protein level was directly downregulated by β-catenin knockdown. β-actin was a loading control. All data are expressed as mean ± S.E.M. Statistical significance was estimated using Student’s t test ( g , i , j , and k ) or one-way ANOVA ( f , h , and l ) from at least three independent experiments; * p

    Journal: Experimental & Molecular Medicine

    Article Title: Wnt signal activation induces midbrain specification through direct binding of the beta-catenin/TCF4 complex to the EN1 promoter in human pluripotent stem cells

    doi: 10.1038/s12276-018-0044-y

    Figure Lengend Snippet: Activation of Wnt signal induces midbrain characteristics in human ESC-derived NPCs. a Efficient induction of neural rosette cells from human ESCs by co-treatment with dorsomorphin and SB431542. b Strong immunoreactivity for SOX1 and Nestin in neural rosette cells. Morphology of neural rosette cells expanding in either the absence ( c ) or the presence of 1 μM BIO ( d ). e NPCs treated with BIO maintained immunoreactivity for SOX1 and Nestin. f Treatment with BIO upregulated EN1 expression and downregulated expressions of BF1 and GBX2 in dose-dependent manner. g BIO treatment significantly increased the number of EN1-positive neural cells. h The inductive effect of BIO treatment on midbrain fate appeared to be more specific than that of FGF8. i Expression pattern of another set of regional markers ( SIX3 for forebrain; PAX2 for midbrain; and HOXA2 for hindbrain) supported the midbrain-biased fate of NPCs treated with BIO. Treatment with other known GSK3 inhibitors, 1-AKP ( j ) and LiCl ( k ), resulted in regionalization comparable to BIO treatment. l Treating NPCs with Wnt antagonists (100 ng/ml DKK-1 and 500 ng/ml frizzled-5) downregulated the endogenous level of EN1 transcript in NPCs. m Immunoblot for β-catenin and EN1 protein after introduction of two different β-catenin-specific shRNAs (shRNA-1 and shRNA-2). EN1 protein level was directly downregulated by β-catenin knockdown. β-actin was a loading control. All data are expressed as mean ± S.E.M. Statistical significance was estimated using Student’s t test ( g , i , j , and k ) or one-way ANOVA ( f , h , and l ) from at least three independent experiments; * p

    Article Snippet: After incubation with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature, the membrane was incubated with primary antibodies (mouse anti-β-catenin (Santa Cruz Biotechnology), mouse anti-EN1 (Abcam, Cambridge, UK), and mouse anti-β-actin (Sigma-Aldrich)) for 1 h at room temperature or overnight at 4 °C.

    Techniques: Activation Assay, Derivative Assay, Expressing, ALP Assay, shRNA

    BACE1 Western blot. (A) Representative Western blot of BACE1 (top images) and beta-actin (bottom images) in MFC (left) and MTC (right). APOE ε3/3 carriers showed higher BACE1 signals than APOE ε4 heterozygotes and homozygotes in ND samples.

    Journal: Current Alzheimer research

    Article Title: BACE1 Levels by APOE Genotype in Non-Demented and Alzheimer\u2019s Post-Mortem Brains

    doi:

    Figure Lengend Snippet: BACE1 Western blot. (A) Representative Western blot of BACE1 (top images) and beta-actin (bottom images) in MFC (left) and MTC (right). APOE ε3/3 carriers showed higher BACE1 signals than APOE ε4 heterozygotes and homozygotes in ND samples.

    Article Snippet: The membranes were then stripped and reprobed with mouse anti-β actin (#A1978; Sigma-Aldrich, St. Louis, MO).

    Techniques: Western Blot

    TET1-CD up-regulates TSGs expression. (A). The SMMC 7721 cells were transiently transfected with either TET1-CD plasmids or TET1-mCD plasmids. Expression of TET1-CD and TET1-mCD proteins was analysed by Western blot, and β-actin was used as an internal control. (B) (C). The SMMC 7721 cells were cultured in the 6 mm plate for 24h and then transiently transfected with either pflag-CMV4 vector (control), TET1-CD or TET1-mCD. Expression of TSGs and oncogenes was analyzed by Quantitative RT-PCR 48h after transient transfection, and the results were represented as mean ± SD of three independent experiments. *p

    Journal: PLoS ONE

    Article Title: Effects of a single transient transfection of Ten-eleven translocation 1 catalytic domain on hepatocellular carcinoma

    doi: 10.1371/journal.pone.0207139

    Figure Lengend Snippet: TET1-CD up-regulates TSGs expression. (A). The SMMC 7721 cells were transiently transfected with either TET1-CD plasmids or TET1-mCD plasmids. Expression of TET1-CD and TET1-mCD proteins was analysed by Western blot, and β-actin was used as an internal control. (B) (C). The SMMC 7721 cells were cultured in the 6 mm plate for 24h and then transiently transfected with either pflag-CMV4 vector (control), TET1-CD or TET1-mCD. Expression of TSGs and oncogenes was analyzed by Quantitative RT-PCR 48h after transient transfection, and the results were represented as mean ± SD of three independent experiments. *p

    Article Snippet: Then, the membrane was blocked with 5% non-fat milk for 2h at room temperature, and then incubated at 4°C overnight with anti-TET1 (GeneTex) and anti-FLAG (Stratagene) antibody (1:1000 dilution), or mouse monoclonal anti-β-actin (Sigma) antibody (1:5000 dilution) for the internal control.

    Techniques: Expressing, Transfection, Western Blot, Cell Culture, Plasmid Preparation, Quantitative RT-PCR

    STAT3 expression in the model of chronic hepatitis B. (A) STAT3 levels in the liver tissues at the indicated time points (two representatives of each), the tumor tissues (18M-Tum) and HCC cell lines (HepG2 and PLC/PRF/5) were evaluated by Western blotting, and normalized to β-actin. (B) The liver tissues were stained with hematoxylin and eosin, and analyzed immunohistochemically. #282 and #283 mice (0M: unmanipuladed) were 24-month-old. Original magnifications, x 200 (bar represents 50 μm).

    Journal: PLoS ONE

    Article Title: Gene expression profiling of hepatocarcinogenesis in a mouse model of chronic hepatitis B

    doi: 10.1371/journal.pone.0185442

    Figure Lengend Snippet: STAT3 expression in the model of chronic hepatitis B. (A) STAT3 levels in the liver tissues at the indicated time points (two representatives of each), the tumor tissues (18M-Tum) and HCC cell lines (HepG2 and PLC/PRF/5) were evaluated by Western blotting, and normalized to β-actin. (B) The liver tissues were stained with hematoxylin and eosin, and analyzed immunohistochemically. #282 and #283 mice (0M: unmanipuladed) were 24-month-old. Original magnifications, x 200 (bar represents 50 μm).

    Article Snippet: [ ] Anti-STAT3 monoclonal (79D7; Cell Signaling), anti-mouse p53 polyclonal (Leica, Newcastle, UK), anti-phospho-p53 (Ser6 and Ser15) (Cell Signaling) and anti-β-actin monoclonal (Sigma–Aldrich, St. Louis, MO) antibodies were used for protein detection.

    Techniques: Expressing, Planar Chromatography, Western Blot, Staining, Mouse Assay

    TP53 expression in the model of chronic hepatitis B. Total TP53 levels and phosphorylated TP53 at serines 6 and 15 (p-p53-Ser 6 and -Ser 15) in the liver tissues at the indicated time points (two representatives of each) and the tumor tissues (18M-Tum) were evaluated by Western blotting, and normalized to β -actin.

    Journal: PLoS ONE

    Article Title: Gene expression profiling of hepatocarcinogenesis in a mouse model of chronic hepatitis B

    doi: 10.1371/journal.pone.0185442

    Figure Lengend Snippet: TP53 expression in the model of chronic hepatitis B. Total TP53 levels and phosphorylated TP53 at serines 6 and 15 (p-p53-Ser 6 and -Ser 15) in the liver tissues at the indicated time points (two representatives of each) and the tumor tissues (18M-Tum) were evaluated by Western blotting, and normalized to β -actin.

    Article Snippet: [ ] Anti-STAT3 monoclonal (79D7; Cell Signaling), anti-mouse p53 polyclonal (Leica, Newcastle, UK), anti-phospho-p53 (Ser6 and Ser15) (Cell Signaling) and anti-β-actin monoclonal (Sigma–Aldrich, St. Louis, MO) antibodies were used for protein detection.

    Techniques: Expressing, Western Blot

    ( A ) Western blot analysis showing changes in the protein expression of cleaved caspase-3 in chemo-resistant HCT-116 cells after 48 h incubation of cells in the absence or presence of 5-Fu + Ox alone or in combination with either CDF or curcumin, β-actin ( loading control ) levels are also shown ( upper panel ), and changes in the levels of 19 kDa and 17 kDa fragments of cleaved caspase-3 relative to control normalized to β-actin, as determined by densitometry analysis which, are depicted in the histogram ( lower panel ). ( B ) Changes in the caspase-3 activity in the chemo-resistant HT-29 cells. Values are mean±SD of 4 experiments. p

    Journal: Pharmaceutical research

    Article Title: Difluorinated-Curcumin (CDF): A Novel Curcumin Analog is a Potent Inhibitor of Colon Cancer Stem-Like Cells

    doi: 10.1007/s11095-010-0336-y

    Figure Lengend Snippet: ( A ) Western blot analysis showing changes in the protein expression of cleaved caspase-3 in chemo-resistant HCT-116 cells after 48 h incubation of cells in the absence or presence of 5-Fu + Ox alone or in combination with either CDF or curcumin, β-actin ( loading control ) levels are also shown ( upper panel ), and changes in the levels of 19 kDa and 17 kDa fragments of cleaved caspase-3 relative to control normalized to β-actin, as determined by densitometry analysis which, are depicted in the histogram ( lower panel ). ( B ) Changes in the caspase-3 activity in the chemo-resistant HT-29 cells. Values are mean±SD of 4 experiments. p

    Article Snippet: Rabbit anti-p-EGF-Receptor (Tyr 1173), rabbit anti-c-Myc, rabbit anti-phospho-β-catenin and rabbit anti-cleaved caspase-3 antibodies were the products of Cell Signaling, Danvers, MA, and the mouse anti-β-actin antibodies were purchased from Chemicon International, Billerica, MA.

    Techniques: Western Blot, Expressing, Incubation, Activity Assay

    Quantitative real-time PCR (qRT-PCR) analysis showing mRNA expression of colon CSC markers CD44 and CD166 ( left panel ) and changes in protein levels of ABCG2, pEGFR-Y 1173 and p-IGF-1R in chemo-resistant HCT-116 cells in response to 5-FU + Ox alone or in combination with either curcumin or CDF ( right panel ); β-actin ( loading control ) levels is also shown. The numbers represent percent of corresponding control normalized to β-actin. * p

    Journal: Pharmaceutical research

    Article Title: Difluorinated-Curcumin (CDF): A Novel Curcumin Analog is a Potent Inhibitor of Colon Cancer Stem-Like Cells

    doi: 10.1007/s11095-010-0336-y

    Figure Lengend Snippet: Quantitative real-time PCR (qRT-PCR) analysis showing mRNA expression of colon CSC markers CD44 and CD166 ( left panel ) and changes in protein levels of ABCG2, pEGFR-Y 1173 and p-IGF-1R in chemo-resistant HCT-116 cells in response to 5-FU + Ox alone or in combination with either curcumin or CDF ( right panel ); β-actin ( loading control ) levels is also shown. The numbers represent percent of corresponding control normalized to β-actin. * p

    Article Snippet: Rabbit anti-p-EGF-Receptor (Tyr 1173), rabbit anti-c-Myc, rabbit anti-phospho-β-catenin and rabbit anti-cleaved caspase-3 antibodies were the products of Cell Signaling, Danvers, MA, and the mouse anti-β-actin antibodies were purchased from Chemicon International, Billerica, MA.

    Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    Effect of 5-FU + Ox alone or in combination with either curcumin or CDF on NF-κB DNA binding activity ( left panel ) and changes in the levels β-catenin, p-β-catenin, Cox-2 and c-Myc, in chemo-resistant HCT-116 cells ( right panel ); the levels of β-actin ( loading control ) are also shown. The numbers represent percent of corresponding control normalized to β-actin. * p

    Journal: Pharmaceutical research

    Article Title: Difluorinated-Curcumin (CDF): A Novel Curcumin Analog is a Potent Inhibitor of Colon Cancer Stem-Like Cells

    doi: 10.1007/s11095-010-0336-y

    Figure Lengend Snippet: Effect of 5-FU + Ox alone or in combination with either curcumin or CDF on NF-κB DNA binding activity ( left panel ) and changes in the levels β-catenin, p-β-catenin, Cox-2 and c-Myc, in chemo-resistant HCT-116 cells ( right panel ); the levels of β-actin ( loading control ) are also shown. The numbers represent percent of corresponding control normalized to β-actin. * p

    Article Snippet: Rabbit anti-p-EGF-Receptor (Tyr 1173), rabbit anti-c-Myc, rabbit anti-phospho-β-catenin and rabbit anti-cleaved caspase-3 antibodies were the products of Cell Signaling, Danvers, MA, and the mouse anti-β-actin antibodies were purchased from Chemicon International, Billerica, MA.

    Techniques: Binding Assay, Activity Assay

    ( A ) Western blot showing changes in the levels of pro-apoptotic Bax, and anti-apoptotic Bcl-xL in untreated parental as compared to chemo-resistant HCT-116 cells in response to the absence or presence of 5-Fu + Ox alone or in combination with either CDF or curcumin. The numbers represent percent of corresponding control normalized to β-actin. ( B ) Induction of early apoptosis as determined by acridine-orange/ethidium-bromide staining in chemo-resistant HCT-116 and HT-29 cells after incubation of cells in the absence or presence of 5-Fu + Ox alone or in combination with either CDF or curcumin. Values are mean±SD of 4 experiments. * p

    Journal: Pharmaceutical research

    Article Title: Difluorinated-Curcumin (CDF): A Novel Curcumin Analog is a Potent Inhibitor of Colon Cancer Stem-Like Cells

    doi: 10.1007/s11095-010-0336-y

    Figure Lengend Snippet: ( A ) Western blot showing changes in the levels of pro-apoptotic Bax, and anti-apoptotic Bcl-xL in untreated parental as compared to chemo-resistant HCT-116 cells in response to the absence or presence of 5-Fu + Ox alone or in combination with either CDF or curcumin. The numbers represent percent of corresponding control normalized to β-actin. ( B ) Induction of early apoptosis as determined by acridine-orange/ethidium-bromide staining in chemo-resistant HCT-116 and HT-29 cells after incubation of cells in the absence or presence of 5-Fu + Ox alone or in combination with either CDF or curcumin. Values are mean±SD of 4 experiments. * p

    Article Snippet: Rabbit anti-p-EGF-Receptor (Tyr 1173), rabbit anti-c-Myc, rabbit anti-phospho-β-catenin and rabbit anti-cleaved caspase-3 antibodies were the products of Cell Signaling, Danvers, MA, and the mouse anti-β-actin antibodies were purchased from Chemicon International, Billerica, MA.

    Techniques: Western Blot, Staining, Incubation

    Increased expression of TGF-β in retina of mice after Laser-induced CNV formation. ( A ) Flat mounts were fluorescently labeled with TGF-β antibody (green) and the nuclear staining with Hoechst (blue). Low levels of TGF-β expression were detected in ganglion cells of retina and choroid of Nor control mice. However, the strong immunoreactivity could be observed in the ganglion cells, outer nuclear layer, inner nuclear layer of retina and choroid at two weeks after Laser injury (Scale bars = 50 μm). ( B ) Typical result of Western blot showed changes of TGF-β protein levels in RPE/choroid/sclera isolated from the mice with or without Laser-induced CNV formation. ( C ) The quantitative analysis results demonstrated that the TGF-β protein levels were significantly up regulated at one week and reached a peak at three weeks in RPE/choroid/sclera of mice after Laser photocoagulation. The β-actin was used as loading control and reference protein, and the ratio of TGF-β/β-actin in Nor mice was set as 100%. *P

    Journal: Scientific Reports

    Article Title: TGF-β participates choroid neovascularization through Smad2/3-VEGF/TNF-α signaling in mice with Laser-induced wet age-related macular degeneration

    doi: 10.1038/s41598-017-10124-4

    Figure Lengend Snippet: Increased expression of TGF-β in retina of mice after Laser-induced CNV formation. ( A ) Flat mounts were fluorescently labeled with TGF-β antibody (green) and the nuclear staining with Hoechst (blue). Low levels of TGF-β expression were detected in ganglion cells of retina and choroid of Nor control mice. However, the strong immunoreactivity could be observed in the ganglion cells, outer nuclear layer, inner nuclear layer of retina and choroid at two weeks after Laser injury (Scale bars = 50 μm). ( B ) Typical result of Western blot showed changes of TGF-β protein levels in RPE/choroid/sclera isolated from the mice with or without Laser-induced CNV formation. ( C ) The quantitative analysis results demonstrated that the TGF-β protein levels were significantly up regulated at one week and reached a peak at three weeks in RPE/choroid/sclera of mice after Laser photocoagulation. The β-actin was used as loading control and reference protein, and the ratio of TGF-β/β-actin in Nor mice was set as 100%. *P

    Article Snippet: Western Blot Analysis Antibodies used in this study are listed as follows: rabbit anti-TGF-β polyclonal antibody (1:500; catalogue number ab66043), rabbit anti-VEGF polyclonal antibody (1:1,000; catalogue number ab46154) and rabbit anti-TNF-α polyclonal antibody (1:500; catalogue number ab9635, Abcam, Cambridge, UK); rabbit anti-Smad2/3 polyclonal antibody (1:1,000; catalogue number #5678) and anti-Phospho-Smad2/3 monoclonal antibody (1:1,000; catalogue number #8828, Cell Signaling Technology, Boston, MA 02241–3843, USA); mouse anti-β-actin monoclonal antibody (1:3,000; Sigma-Aldrich Corp. St. Louis, MO 63103, USA); and the horseradish peroxidase conjugated goat anti-rabbit or anti-mouse IgG as secondary antibody (1:4,000; Stressgen Biotechnologies Corporation, Victoria BC, Canada).

    Techniques: Expressing, Mouse Assay, Labeling, Staining, Western Blot, Isolation

    In vitro Trypanosoma cruzi infection induces expression of Wnt proteins, Frizzled (Fzd) receptors, and β-catenin macrophages. Bone marrow-derived macrophages were in vitro infected with T. cruzi trypomastigotes or left uninfected (NI) and then evaluated for expression of Wnt proteins and Fzd receptors at different times post-infection (pi). (A) Expression of Wnt3a and Wnt5a mRNA (relative to β-actin) was determined by quantitative real-time-PCR. (B) The relative abundance of Wnt3, Wnt5a, and β-actin in the cell lysates were determined by Western blot and densitometry at 12 h pi. Representative Western blot and the ratio of Wnt3a or Wnt5a to β-actin are shown. (C) Expression of Fzd4, Fzd8, Fzd9, and Fzd6 mRNA (relative to β-actin) was determined by q-PCR. The results are expressed as the average of three independent experiments ± SEM. Abbreviation: AU, arbitrary units (* P

    Journal: Frontiers in Immunology

    Article Title: Trypanosoma cruzi Exploits Wnt Signaling Pathway to Promote Its Intracellular Replication in Macrophages

    doi: 10.3389/fimmu.2018.00859

    Figure Lengend Snippet: In vitro Trypanosoma cruzi infection induces expression of Wnt proteins, Frizzled (Fzd) receptors, and β-catenin macrophages. Bone marrow-derived macrophages were in vitro infected with T. cruzi trypomastigotes or left uninfected (NI) and then evaluated for expression of Wnt proteins and Fzd receptors at different times post-infection (pi). (A) Expression of Wnt3a and Wnt5a mRNA (relative to β-actin) was determined by quantitative real-time-PCR. (B) The relative abundance of Wnt3, Wnt5a, and β-actin in the cell lysates were determined by Western blot and densitometry at 12 h pi. Representative Western blot and the ratio of Wnt3a or Wnt5a to β-actin are shown. (C) Expression of Fzd4, Fzd8, Fzd9, and Fzd6 mRNA (relative to β-actin) was determined by q-PCR. The results are expressed as the average of three independent experiments ± SEM. Abbreviation: AU, arbitrary units (* P

    Article Snippet: Anti-β-actin (mAbcam8226, Abcam) was used as loading control.

    Techniques: In Vitro, Infection, Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Polymerase Chain Reaction

    Trypanosoma cruzi infection induces early β-catenin activation in macrophages. Bone marrow-derived macrophages were in vitro infected with trypomastigotes of T. cruzi or left uninfected (NI) and then evaluated for β-catenin activation. (A) mRNA and protein expression levels of β-catenin by q-PCR and Western blot at different times post-infection (pi). β-Catenin mRNA relative to β-actin is shown. A representative Western blot and the ratio of β-catenin to β-actin are shown. The results are expressed as the average of three independent experiments ± SEM. Abbreviation: AU, arbitrary units. (B) Expression and localization of β-catenin by immunofluorescence and confocal microscopy. (C) mRNA relative expression of β-catenin target genes Axin1, Wisp1, and Ccnd1 by q-PCR. mRNA expression levels normalized over the expression of β-actin are expressed as the average of three independent experiments ± SEM. (D) Peritoneal macrophages were obtained from uninfected or infected mice at 24 h pi, and the levels of expression and localization of β-catenin were evaluated by immunofluorescence and confocal microscopy. In panels (B,D) , a representative field for each group is shown [ (B,D) .” Green, β-catenin; red, DAPI (* P

    Journal: Frontiers in Immunology

    Article Title: Trypanosoma cruzi Exploits Wnt Signaling Pathway to Promote Its Intracellular Replication in Macrophages

    doi: 10.3389/fimmu.2018.00859

    Figure Lengend Snippet: Trypanosoma cruzi infection induces early β-catenin activation in macrophages. Bone marrow-derived macrophages were in vitro infected with trypomastigotes of T. cruzi or left uninfected (NI) and then evaluated for β-catenin activation. (A) mRNA and protein expression levels of β-catenin by q-PCR and Western blot at different times post-infection (pi). β-Catenin mRNA relative to β-actin is shown. A representative Western blot and the ratio of β-catenin to β-actin are shown. The results are expressed as the average of three independent experiments ± SEM. Abbreviation: AU, arbitrary units. (B) Expression and localization of β-catenin by immunofluorescence and confocal microscopy. (C) mRNA relative expression of β-catenin target genes Axin1, Wisp1, and Ccnd1 by q-PCR. mRNA expression levels normalized over the expression of β-actin are expressed as the average of three independent experiments ± SEM. (D) Peritoneal macrophages were obtained from uninfected or infected mice at 24 h pi, and the levels of expression and localization of β-catenin were evaluated by immunofluorescence and confocal microscopy. In panels (B,D) , a representative field for each group is shown [ (B,D) .” Green, β-catenin; red, DAPI (* P

    Article Snippet: Anti-β-actin (mAbcam8226, Abcam) was used as loading control.

    Techniques: Infection, Activation Assay, Derivative Assay, In Vitro, Expressing, Polymerase Chain Reaction, Western Blot, Immunofluorescence, Confocal Microscopy, Mouse Assay

    Trypanosoma cruzi infection induces late Wnt/Ca +2 pathway activation in macrophages. Bone marrow-derived macrophages were in vitro infected with trypomastigotes (Tps) of T. cruzi or left uninfected (NI) and then evaluated for calcium/calmodulin-dependent kinase II (CaMKII) phosphorylated at Thr286 expression and NFATc1 expression and localization at different times post-infection (pi). Phosphorylated CAMKII (p-CAMKII) expression (A) and NFATc1 expression (B) were assessed by Western blot and normalized to β-actin. (C) .” Green, NFATc1; red, DAPI. (D,E) Bone marrow-derived macrophages (BMM) were treated for 24 h with the PORCN inhibitor (IWP-L6) or left untreated (control), infected with T. cruzi Tps and assayed for p-CaMKII and NFATc1 expression at 24 h pi. Ionomycin-activated BMM (20 min, 1 µM) were used as a positive control. In panels (A,B,D,E) , a representative Western blot and the ratio of protein expression to β-actin are shown. The results are expressed as the average of three independent experiments ± SEM. Abbreviation: AU, arbitrary units (* P

    Journal: Frontiers in Immunology

    Article Title: Trypanosoma cruzi Exploits Wnt Signaling Pathway to Promote Its Intracellular Replication in Macrophages

    doi: 10.3389/fimmu.2018.00859

    Figure Lengend Snippet: Trypanosoma cruzi infection induces late Wnt/Ca +2 pathway activation in macrophages. Bone marrow-derived macrophages were in vitro infected with trypomastigotes (Tps) of T. cruzi or left uninfected (NI) and then evaluated for calcium/calmodulin-dependent kinase II (CaMKII) phosphorylated at Thr286 expression and NFATc1 expression and localization at different times post-infection (pi). Phosphorylated CAMKII (p-CAMKII) expression (A) and NFATc1 expression (B) were assessed by Western blot and normalized to β-actin. (C) .” Green, NFATc1; red, DAPI. (D,E) Bone marrow-derived macrophages (BMM) were treated for 24 h with the PORCN inhibitor (IWP-L6) or left untreated (control), infected with T. cruzi Tps and assayed for p-CaMKII and NFATc1 expression at 24 h pi. Ionomycin-activated BMM (20 min, 1 µM) were used as a positive control. In panels (A,B,D,E) , a representative Western blot and the ratio of protein expression to β-actin are shown. The results are expressed as the average of three independent experiments ± SEM. Abbreviation: AU, arbitrary units (* P

    Article Snippet: Anti-β-actin (mAbcam8226, Abcam) was used as loading control.

    Techniques: Infection, Activation Assay, Derivative Assay, In Vitro, Expressing, Western Blot, Positive Control

    Experimental Trypanosoma cruzi infection induces the expression of Wnt pathway proteins. Western blot analysis of Wnt3a, Wnt5a, and β-catenin expression in spleen mononuclear cell homogenates derived from uninfected (NI) or T. cruzi -infected B6 mice at different times post-infection. Images show one representative NI and one infected mice by time point. Each bar represents the mean ± SEM of protein relative expression levels quantified by scanning the intensity of bands areas in the homogenates normalized to β-actin ( n = 5 mice per time point) (* P

    Journal: Frontiers in Immunology

    Article Title: Trypanosoma cruzi Exploits Wnt Signaling Pathway to Promote Its Intracellular Replication in Macrophages

    doi: 10.3389/fimmu.2018.00859

    Figure Lengend Snippet: Experimental Trypanosoma cruzi infection induces the expression of Wnt pathway proteins. Western blot analysis of Wnt3a, Wnt5a, and β-catenin expression in spleen mononuclear cell homogenates derived from uninfected (NI) or T. cruzi -infected B6 mice at different times post-infection. Images show one representative NI and one infected mice by time point. Each bar represents the mean ± SEM of protein relative expression levels quantified by scanning the intensity of bands areas in the homogenates normalized to β-actin ( n = 5 mice per time point) (* P

    Article Snippet: Anti-β-actin (mAbcam8226, Abcam) was used as loading control.

    Techniques: Infection, Expressing, Western Blot, Derivative Assay, Mouse Assay

    Reduced PAX8 expression leads to decreased expression of BCL2 and WT1. (A) The PAX8 -knockdown (siPAX8) in the A172 glioma cell line by siRNA (PAX8-1) produced a reduction in the BCL2 expression levels. Cells lysates were prepared 36 hours after siRNA transfection, and the PAX8, BCL2, p53, and β-actin (loading control) expression levels were measured by western blot. For controls, A172 cells were transfected with mock-treated (Moc), non-targeting siRNAs (NT1, NT2, and NT3) and scrambled s8-1 siRNA (scPAX8). To ensure the reduction in the glioma cell growth rate associated with the PAX8 -knockdown was not due to p53 function, p53 was also knocked down in A172 cells (sip53) independently or in combination with a PAX8 siRNA (siPAX8 p53). (B ) The PAX8 -knockdown (siPAX8) in the A172 glioma cell line by siRNA (PAX8-1) produced a reduction in the WT1 expression levels. (C) The BCL2 -knockdown produced a similar reduction in the cell growth rate compared to PAX8 -knockdown in the A172 glioma cell line. Cells were transfected with a BCL2 siRNA (siBCL2) or a PAX8 siRNA (PAX8-1, siPAX8). For controls, A172 cells were mock-transfected (Moc) or transfected with non-targeting siRNAs (NT1 and NT3). The percentage of live cells was determined by the trypan blue exclusion assay every 24 hours post-transfection. ( Insert ) Western blotting shows the BCL2-knockdown with a BCL2 siRNA and no BCL2-knockdown in controls; the loading control is β-actin.

    Journal: BMC Cancer

    Article Title: Increased paired box transcription factor 8 has a survival function in Glioma

    doi: 10.1186/1471-2407-14-159

    Figure Lengend Snippet: Reduced PAX8 expression leads to decreased expression of BCL2 and WT1. (A) The PAX8 -knockdown (siPAX8) in the A172 glioma cell line by siRNA (PAX8-1) produced a reduction in the BCL2 expression levels. Cells lysates were prepared 36 hours after siRNA transfection, and the PAX8, BCL2, p53, and β-actin (loading control) expression levels were measured by western blot. For controls, A172 cells were transfected with mock-treated (Moc), non-targeting siRNAs (NT1, NT2, and NT3) and scrambled s8-1 siRNA (scPAX8). To ensure the reduction in the glioma cell growth rate associated with the PAX8 -knockdown was not due to p53 function, p53 was also knocked down in A172 cells (sip53) independently or in combination with a PAX8 siRNA (siPAX8 p53). (B ) The PAX8 -knockdown (siPAX8) in the A172 glioma cell line by siRNA (PAX8-1) produced a reduction in the WT1 expression levels. (C) The BCL2 -knockdown produced a similar reduction in the cell growth rate compared to PAX8 -knockdown in the A172 glioma cell line. Cells were transfected with a BCL2 siRNA (siBCL2) or a PAX8 siRNA (PAX8-1, siPAX8). For controls, A172 cells were mock-transfected (Moc) or transfected with non-targeting siRNAs (NT1 and NT3). The percentage of live cells was determined by the trypan blue exclusion assay every 24 hours post-transfection. ( Insert ) Western blotting shows the BCL2-knockdown with a BCL2 siRNA and no BCL2-knockdown in controls; the loading control is β-actin.

    Article Snippet: Blots were probed with primary antibodies raised against PAX8 (MRQ-50, Cell Marque), Bcl-2 (Clone 124, Dako), p53 (1C12, Cell Signaling Technology, Beverly, MA), WT1 (6FH2, Dako, Glostrup, Denmark) and β-actin (AC-15, Abcam, UK) according to the manufacturers’ instruction, or that optimised in the current study (1:200 dilution for WT1).

    Techniques: Expressing, Produced, Transfection, Western Blot, Trypan Blue Exclusion Assay

    MUC1 is widely overexpressed in NSCLC cells, correlating with STAT3 activation. (A) Protein expression of MUC1-C and total and Tyr705 phosphorylated STAT3 levels were determined in 14 human NSCLC cell lines by Western blot analyses. Equal loading and transfer were shown by repeat probing with β-actin. (B) mRNA levels of MUC1 in a subset of cells were evaluated with real-time RT-PCR. MUC1 mRNA expression levels in each cell line were normalized to GAPDH mRNA, with expression in A549 cells set to an arbitrary value of 1. * P

    Journal: International journal of oncology

    Article Title: MUC1 is a downstream target of STAT3 and regulates lung cancer cell survival and invasion

    doi:

    Figure Lengend Snippet: MUC1 is widely overexpressed in NSCLC cells, correlating with STAT3 activation. (A) Protein expression of MUC1-C and total and Tyr705 phosphorylated STAT3 levels were determined in 14 human NSCLC cell lines by Western blot analyses. Equal loading and transfer were shown by repeat probing with β-actin. (B) mRNA levels of MUC1 in a subset of cells were evaluated with real-time RT-PCR. MUC1 mRNA expression levels in each cell line were normalized to GAPDH mRNA, with expression in A549 cells set to an arbitrary value of 1. * P

    Article Snippet: Anti-β-actin and donkey anti-rabbit IgG HRP-conjugated secondary antibody were purchased from Sigma (St. Louis, MO) and Amersham Biosciences (Piscataway, NJ), respectively.

    Techniques: Activation Assay, Expressing, Western Blot, Quantitative RT-PCR

    Effects of MUC1 knockdown or overexpression on multiple cell signals in NSCLC cells. (A) H358, HCC827, and H441 cells were exposed to siRNA for 72 h, and (B) A549 cells were transfected with MUC1-C plasmid for 48 h followed by measurement of phosphorylated tyrosine STAT3, Akt, Src, FAK, and apoptotic-related proteins by Western blot analysis. Equal loading and transfer were shown by repeat probing with β-actin.

    Journal: International journal of oncology

    Article Title: MUC1 is a downstream target of STAT3 and regulates lung cancer cell survival and invasion

    doi:

    Figure Lengend Snippet: Effects of MUC1 knockdown or overexpression on multiple cell signals in NSCLC cells. (A) H358, HCC827, and H441 cells were exposed to siRNA for 72 h, and (B) A549 cells were transfected with MUC1-C plasmid for 48 h followed by measurement of phosphorylated tyrosine STAT3, Akt, Src, FAK, and apoptotic-related proteins by Western blot analysis. Equal loading and transfer were shown by repeat probing with β-actin.

    Article Snippet: Anti-β-actin and donkey anti-rabbit IgG HRP-conjugated secondary antibody were purchased from Sigma (St. Louis, MO) and Amersham Biosciences (Piscataway, NJ), respectively.

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

    Increased expression of arterial Ca 2+ transport proteins following a 14 day icv Ang II infusion. Panels A , B and C , representative Western blots and summary data for NCX1, TRPC6 and SERCA2 expression, respectively, in de-endothelialized aorta. Blots were normalized for β-actin as a loading control and the changes shown are relative to the control group. All lanes were loaded with 10–20 µg of membrane protein. The expected molecular weights are: NCX1, 116 KDa; TRPC6, 111 KDa, SERCA2 115 KDA, and β-actin, 42 KDa. C = vehicle control; A = Ang II; A+E = Ang II + eplerenone; A+F = Ang II + FAD-286; *P

    Journal: PLoS ONE

    Article Title: Neuroendocrine Humoral and Vascular Components in the Pressor Pathway for Brain Angiotensin II: A New Axis in Long Term Blood Pressure Control

    doi: 10.1371/journal.pone.0108916

    Figure Lengend Snippet: Increased expression of arterial Ca 2+ transport proteins following a 14 day icv Ang II infusion. Panels A , B and C , representative Western blots and summary data for NCX1, TRPC6 and SERCA2 expression, respectively, in de-endothelialized aorta. Blots were normalized for β-actin as a loading control and the changes shown are relative to the control group. All lanes were loaded with 10–20 µg of membrane protein. The expected molecular weights are: NCX1, 116 KDa; TRPC6, 111 KDa, SERCA2 115 KDA, and β-actin, 42 KDa. C = vehicle control; A = Ang II; A+E = Ang II + eplerenone; A+F = Ang II + FAD-286; *P

    Article Snippet: Gel loading was controlled with monoclonal anti-β-actin antibodies (dilution 1∶10,000; Sigma-Aldrich).

    Techniques: Expressing, Western Blot

    The 3′-UTR of ECE-1 represses ECE-1 protein expression. PC-3, RWPE-1, Huh7 and CHO cells were transiently transfected with empty vector (1), V5-tagged ECE-1c cDNA (2) or V5-tagged ECE-1c cDNA with full-length 3′ UTR (3) and immunoblotted for V5 (A). β-actin was used as a loading control. The PC-3 blot was reprobed for ECE-1 expression. Data represent the ratio of V5:actin expression ± S.E.M. (B; n = 3, * = P

    Journal: PLoS ONE

    Article Title: Endothelin-Converting Enzyme-1 (ECE-1) Is Post-Transcriptionally Regulated by Alternative Polyadenylation

    doi: 10.1371/journal.pone.0083260

    Figure Lengend Snippet: The 3′-UTR of ECE-1 represses ECE-1 protein expression. PC-3, RWPE-1, Huh7 and CHO cells were transiently transfected with empty vector (1), V5-tagged ECE-1c cDNA (2) or V5-tagged ECE-1c cDNA with full-length 3′ UTR (3) and immunoblotted for V5 (A). β-actin was used as a loading control. The PC-3 blot was reprobed for ECE-1 expression. Data represent the ratio of V5:actin expression ± S.E.M. (B; n = 3, * = P

    Article Snippet: The following antibodies were used at the following dilutions: monoclonal anti-V5 antibody 1∶500 (Invitrogen), goat polyclonal anti-ECE-1 1:1000 (R & D Systems), rabbit polyclonal anti-Upf1 1:200 (Santa Cruz) and monoclonal anti-β-actin 1∶10,000 (Sigma).

    Techniques: Expressing, Transfection, Plasmid Preparation

    Protein expression levels of PERK, IRE1α and ATF6 in ovaries of aged mice. Representative western blot images (a) and quantification of PERK, IRE1α and ATF6 protein expression levels (b). β-ACTIN served as an internal control. Data are expressed as the mean ± SEM, expressed by error bars.

    Journal: The Journal of Veterinary Medical Science

    Article Title: Intravenous human endothelial progenitor cell administration into aged mice enhances embryo development and oocyte quality by reducing inflammation, endoplasmic reticulum stress and apoptosis

    doi: 10.1292/jvms.18-0242

    Figure Lengend Snippet: Protein expression levels of PERK, IRE1α and ATF6 in ovaries of aged mice. Representative western blot images (a) and quantification of PERK, IRE1α and ATF6 protein expression levels (b). β-ACTIN served as an internal control. Data are expressed as the mean ± SEM, expressed by error bars.

    Article Snippet: The loading and blotting of the amount of protein was verified by reprobing the membrane with anti-β actin antibody (1:5,000; Abcam) followed by Coomassie Blue staining.

    Techniques: Expressing, Mouse Assay, Western Blot

    Gastrodin (GSTD) increased osteogenic transcription Runx2, decreased adipogenic factor peroxisome proliferator-activated receptor (PPAR)γ isoform 2 and activated nuclear factor-like 2 (NRF2) pathway protein expression levels. Representative western blot analyses for total protein was analyzed by western blotting in dexamethasone (DEX)-treated MC3T3-E1 cells with or without GSTD pretreatment at different doses. The relative protein expression levels were measured using the fold-change in each protein relative to β-actin from the same sample. The data are expressed as the mean ± standard deviation. ## P

    Journal: International Journal of Molecular Medicine

    Article Title: Gastrodin protects MC3T3-E1 osteoblasts from dexamethasone-induced cellular dysfunction and promotes bone formation via induction of the NRF2 signaling pathway

    doi: 10.3892/ijmm.2018.3414

    Figure Lengend Snippet: Gastrodin (GSTD) increased osteogenic transcription Runx2, decreased adipogenic factor peroxisome proliferator-activated receptor (PPAR)γ isoform 2 and activated nuclear factor-like 2 (NRF2) pathway protein expression levels. Representative western blot analyses for total protein was analyzed by western blotting in dexamethasone (DEX)-treated MC3T3-E1 cells with or without GSTD pretreatment at different doses. The relative protein expression levels were measured using the fold-change in each protein relative to β-actin from the same sample. The data are expressed as the mean ± standard deviation. ## P

    Article Snippet: Rabbit anti-HO-1 (cat. no. ab68477; 1:1,000), rabbit anti-NQO-1 (cat. no. ab76956; 1:1,000), and mouse anti-β-actin (cat. no. ab8245; 1:1,000), Anti-OCN (cat. no. ab93876; 1:1,000) monoclonal antibodies were purchased from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Western Blot, Standard Deviation

    ORAIs and STIMs in human kidney and regulation under diabetic condition. a Immunostaining for ORAIs in normal and diabetic kidney tissue sections. The diabetic kidney sections showing typical mesangial expansion and accumulation of mesangial matrix material. Scale bar, 100 µm. b , c Mean ± s.e.m. for the staining intensity (arbitrary unit) in proximal tubules and distal tubules, respectively. The average of nine staining fields for each patient was calculated for proximal or distal tubule staining ( n = 6 for normal kidney, n = 8 for diabetic kidney). Also see staining for STIM1 and STIM2 (Supplementary Fig. 3 ). d Primary cultured human proximal tubular epithelial cells (PTECs) were characterized by lectin staining (red). Scale bars, 50 µm. e PTECs were cultured with normal (5.5 mM) and high (25 mM) glucose for 60 h. ORAI proteins were detected by western blotting ( n = 6). f The mRNA of ORAIs was quantified by real-time PCR. The mean data were from 2–3 independent experiments ( n = 6). g The proximal tubular epithelial cells (HK-2) were treated with or without (control) insulin (10 nM) for 48 h and the mRNA was detected by real-time PCR ( n = 6). h HK-2 cells incubated with tyrosine kinase inhibitor tyrphostin A23 (30 µM) for 48 h ( n = 6). i STIM1 and STIM2 expression after insulin (10 nM) and tyrphostin A23 (30 µM) treatment for 48 h ( n = 9). The β-actin was used as control for relative quantification of mRNA or protein. For PCR experiments, triplicate reactions were set for each gene. The averaged data are displayed as mean ± s.e.m. and the data in e – i are normalized to control. The data sets are compared by t test. Statistical significance is indicated by * P

    Journal: Nature Communications

    Article Title: ORAI channels are critical for receptor-mediated endocytosis of albumin

    doi: 10.1038/s41467-017-02094-y

    Figure Lengend Snippet: ORAIs and STIMs in human kidney and regulation under diabetic condition. a Immunostaining for ORAIs in normal and diabetic kidney tissue sections. The diabetic kidney sections showing typical mesangial expansion and accumulation of mesangial matrix material. Scale bar, 100 µm. b , c Mean ± s.e.m. for the staining intensity (arbitrary unit) in proximal tubules and distal tubules, respectively. The average of nine staining fields for each patient was calculated for proximal or distal tubule staining ( n = 6 for normal kidney, n = 8 for diabetic kidney). Also see staining for STIM1 and STIM2 (Supplementary Fig. 3 ). d Primary cultured human proximal tubular epithelial cells (PTECs) were characterized by lectin staining (red). Scale bars, 50 µm. e PTECs were cultured with normal (5.5 mM) and high (25 mM) glucose for 60 h. ORAI proteins were detected by western blotting ( n = 6). f The mRNA of ORAIs was quantified by real-time PCR. The mean data were from 2–3 independent experiments ( n = 6). g The proximal tubular epithelial cells (HK-2) were treated with or without (control) insulin (10 nM) for 48 h and the mRNA was detected by real-time PCR ( n = 6). h HK-2 cells incubated with tyrosine kinase inhibitor tyrphostin A23 (30 µM) for 48 h ( n = 6). i STIM1 and STIM2 expression after insulin (10 nM) and tyrphostin A23 (30 µM) treatment for 48 h ( n = 9). The β-actin was used as control for relative quantification of mRNA or protein. For PCR experiments, triplicate reactions were set for each gene. The averaged data are displayed as mean ± s.e.m. and the data in e – i are normalized to control. The data sets are compared by t test. Statistical significance is indicated by * P

    Article Snippet: Rabbit anti-β-actin (1:2000 dilution) (SAB4301137, Sigma) was used as an internal standard for protein quantification.

    Techniques: Immunostaining, Staining, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Incubation, Expressing, Polymerase Chain Reaction