β-actin antibody Search Results


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  • 99
    Thermo Fisher β actin
    Overexpression of StAR on retinoid responsive macrophage cholesterol efflux. RAW 264.7 macrophages were transiently transfected with either pCMV5 or pCMV5-StAR expression plasmid, as described under Section 2 . Representative immunoblots illustrate expression of StAR and <t>β-actin</t> in pCMV5 and pCMV5-StAR transfected groups (A). Following 24 h of transfection, macrophages were labeled with 3 H-cholesterol for additional 24 h and then treated with increasing doses of either atRA or 9-cis RA (0–30 μM), for 12 h, in the presence of Apo-A1 (20 μg/ml), as indicated. Cholesterol efflux was determined following the assay described under Section 2 (B). Immunoblots shown are representative of 3–5 independent experiments. β-actin expression was assessed as a loading control. Data represent the mean±SE of four independent experiments. *, p
    β Actin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 70357 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 39990 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 50954 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam β actin
    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 <t>β-actin</t> (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.
    β Actin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 25356 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 12044 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore β actin antibody
    Antibody targeting of sAPP-α promotes amyloidogenic APP processing in vivo . PSAPP mice (3 female mice per group) at 8 months of age were intracerebroventricular (i.c.v.) injected with 2B3 antibody or isotype-matched IgG 2b control antibody at 5 μg/mL and sacrificed at 48 h after the injection. ( a ) Mouse brain homogenates were prepared from these mice and subjected to IB analysis for APP processing. IB using 6E10 antibody shows total APP and three bands corresponding to β-CTF and soluble Aβ species 38 and 40. Densitometry analysis shows the ratios of Aβ 40 to <t>β-actin</t> ( b ) and β-CTF to β-actin ( c ). A t test revealed significant differences in either ratio of Aβ 40 to β-actin or β-CTF to β-actin between 2B3 antibody- and control IgG 2b antibody-i.c.v. injected PSAPP mice. (*** P
    β Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5395 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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    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


    Overexpression of StAR on retinoid responsive macrophage cholesterol efflux. RAW 264.7 macrophages were transiently transfected with either pCMV5 or pCMV5-StAR expression plasmid, as described under Section 2 . Representative immunoblots illustrate expression of StAR and β-actin in pCMV5 and pCMV5-StAR transfected groups (A). Following 24 h of transfection, macrophages were labeled with 3 H-cholesterol for additional 24 h and then treated with increasing doses of either atRA or 9-cis RA (0–30 μM), for 12 h, in the presence of Apo-A1 (20 μg/ml), as indicated. Cholesterol efflux was determined following the assay described under Section 2 (B). Immunoblots shown are representative of 3–5 independent experiments. β-actin expression was assessed as a loading control. Data represent the mean±SE of four independent experiments. *, p

    Journal: Data in Brief

    Article Title: Retinoid regulated macrophage cholesterol efflux involves the steroidogenic acute regulatory protein

    doi: 10.1016/j.dib.2016.03.055

    Figure Lengend Snippet: Overexpression of StAR on retinoid responsive macrophage cholesterol efflux. RAW 264.7 macrophages were transiently transfected with either pCMV5 or pCMV5-StAR expression plasmid, as described under Section 2 . Representative immunoblots illustrate expression of StAR and β-actin in pCMV5 and pCMV5-StAR transfected groups (A). Following 24 h of transfection, macrophages were labeled with 3 H-cholesterol for additional 24 h and then treated with increasing doses of either atRA or 9-cis RA (0–30 μM), for 12 h, in the presence of Apo-A1 (20 μg/ml), as indicated. Cholesterol efflux was determined following the assay described under Section 2 (B). Immunoblots shown are representative of 3–5 independent experiments. β-actin expression was assessed as a loading control. Data represent the mean±SE of four independent experiments. *, p

    Article Snippet: After electrophoresis, the proteins were electrophoretically transferred onto Immuno-Blot PVDF membranes (Bio-Rad Laboratories), which were probed with specific antibodies that recognize StAR and β-actin (Applied Biosystems/Ambion, Austin, TX) , .

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

    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

    BK channels are detected in mitochondrial fractions from normal rat kidney proximal tubular epithelial (NRK) cells. Western blot shows expression of the pore-forming BKα subunit in mitochondrial fractions ( a ) and cytosolic fractions ( c ) from control NRK cells and after exposure to cold storage and rewarming (CS + RW). Manganese superoxide dismutase (MnSOD) served as a mitochondrial marker and loading control for mitochondrial fractions. Proteasome subunit beta type-5 (PSMB5) was used as a cytosolic marker and β-actin was used as a standard loading control. Representative blots are shown using n = 3, where each lane is loaded with 25 μg protein corresponding to a separate experiment. Densitometry analyses for the mitochondrial ( b ) and cytosolic ( d ) fractions are next to corresponding blots; densitometry calculated from two separate blots with a total n = 6; no significant differences detected using p

    Journal: Biomolecules

    Article Title: Specific BK Channel Activator NS11021 Protects Rat Renal Proximal Tubular Cells from Cold Storage—Induced Mitochondrial Injury In Vitro

    doi: 10.3390/biom9120825

    Figure Lengend Snippet: BK channels are detected in mitochondrial fractions from normal rat kidney proximal tubular epithelial (NRK) cells. Western blot shows expression of the pore-forming BKα subunit in mitochondrial fractions ( a ) and cytosolic fractions ( c ) from control NRK cells and after exposure to cold storage and rewarming (CS + RW). Manganese superoxide dismutase (MnSOD) served as a mitochondrial marker and loading control for mitochondrial fractions. Proteasome subunit beta type-5 (PSMB5) was used as a cytosolic marker and β-actin was used as a standard loading control. Representative blots are shown using n = 3, where each lane is loaded with 25 μg protein corresponding to a separate experiment. Densitometry analyses for the mitochondrial ( b ) and cytosolic ( d ) fractions are next to corresponding blots; densitometry calculated from two separate blots with a total n = 6; no significant differences detected using p

    Article Snippet: Western blotting was performed using antibodies against the following proteins: the pore-forming subunit of the BK channel, BKα (1:1000; Alomone Labs, Jerusalem, Israel, #APC-107), manganese superoxide dismutase, MnSOD (1:1000; EMD Millipore, Burlington, MA, USA, #06–984), proteasome subunit beta type-5, PSMB5 (1:1000; Abcam, Cambridge, UK, #ab3330), and β-actin (1:1000; Sigma, #A5441).

    Techniques: Western Blot, Expressing, Marker

    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

    Effects of NSF in a DSS-induced colitis mouse model. (A, B) Mice were orally treated with KRG (200 mg/kg), NSF (200 mg/kg), or SF (200 mg/kg) once a day along with 3% DSS in tap water for seven days. After sacrifice of the mice, colon lengths were measured using a ruler. (C) MPO activity as an indicator of neutrophil infiltration in the stomach was analyzed in total lysates of colon. (D) Body weight increase was determined by changes in body weight after oral administration of NSF. (E) Colon weight per length ratio was calculated by measured colon weight and length scales. (F) Protein levels of COX-1, ZO-1, occludin, and β-actin in the colons of DSS-induced colitis mice treated with NSF were determined by immunoblotting. # p

    Journal: Journal of Ginseng Research

    Article Title: Gastroprotective effects of the nonsaponin fraction of Korean Red Ginseng through cyclooxygenase-1 upregulation

    doi: 10.1016/j.jgr.2019.11.001

    Figure Lengend Snippet: Effects of NSF in a DSS-induced colitis mouse model. (A, B) Mice were orally treated with KRG (200 mg/kg), NSF (200 mg/kg), or SF (200 mg/kg) once a day along with 3% DSS in tap water for seven days. After sacrifice of the mice, colon lengths were measured using a ruler. (C) MPO activity as an indicator of neutrophil infiltration in the stomach was analyzed in total lysates of colon. (D) Body weight increase was determined by changes in body weight after oral administration of NSF. (E) Colon weight per length ratio was calculated by measured colon weight and length scales. (F) Protein levels of COX-1, ZO-1, occludin, and β-actin in the colons of DSS-induced colitis mice treated with NSF were determined by immunoblotting. # p

    Article Snippet: COX-1 and β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Mouse Assay, Activity Assay

    Effects of NSF on cell viability and COX-1 expression in RAW264.7 cells. (A) RAW264.7 cells were treated with NSF (0-200 μg/ml) for 24 h, and cell viability was determined by MTT assay. (B) RAW264.7 cells were treated with NSF (0-200 μg/ml) for 24 h, and the mRNA levels of COX-1 and GAPDH were determined by RT-PCR. (C) Levels of COX-1 and β-actin in whole cell lysates of RAW264.7 cells treated with NSF were determined by immunoblotting. Band intensity was measured by ImageJ. COX-1, cyclooxygenase-1; GAPDH, glyceradehyde-3-phosphate dehydrogenase; NSF, nonsaponin fraction; RT-PCR, reverse transcription polymerase chain reaction.

    Journal: Journal of Ginseng Research

    Article Title: Gastroprotective effects of the nonsaponin fraction of Korean Red Ginseng through cyclooxygenase-1 upregulation

    doi: 10.1016/j.jgr.2019.11.001

    Figure Lengend Snippet: Effects of NSF on cell viability and COX-1 expression in RAW264.7 cells. (A) RAW264.7 cells were treated with NSF (0-200 μg/ml) for 24 h, and cell viability was determined by MTT assay. (B) RAW264.7 cells were treated with NSF (0-200 μg/ml) for 24 h, and the mRNA levels of COX-1 and GAPDH were determined by RT-PCR. (C) Levels of COX-1 and β-actin in whole cell lysates of RAW264.7 cells treated with NSF were determined by immunoblotting. Band intensity was measured by ImageJ. COX-1, cyclooxygenase-1; GAPDH, glyceradehyde-3-phosphate dehydrogenase; NSF, nonsaponin fraction; RT-PCR, reverse transcription polymerase chain reaction.

    Article Snippet: COX-1 and β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Expressing, MTT Assay, Reverse Transcription Polymerase Chain Reaction

    Effects of NSF in an indomethacin-induced gastritis rat model. (A, B) SD rats were orally treated with NSF (100 or 200 mg/kg) or omeprazole (10 mg/kg) once a day along with indomethacin for five days. After sacrifice of the rats, gastric lesions were imaged using an optical digital camera. Formation of stomach lesions was evaluated using a pixel counter. (C) The pH of collected gastric juice was measured by a pH meter. (D, E) Histological examination of sections of gastric tissue stained with hematoxylin and eosin. Images were captured using an optical digital camera. Thicknesses of gastric walls were measured using ImageJ. (F) COX-1 mRNA level was determined by real-time PCR. (G) Protein levels of COX-1 and β-actin were determined by immunoblotting. (H) MPO activity as an indicator of neutrophil infiltration in the stomach was analyzed in total lysates of stomach. # p

    Journal: Journal of Ginseng Research

    Article Title: Gastroprotective effects of the nonsaponin fraction of Korean Red Ginseng through cyclooxygenase-1 upregulation

    doi: 10.1016/j.jgr.2019.11.001

    Figure Lengend Snippet: Effects of NSF in an indomethacin-induced gastritis rat model. (A, B) SD rats were orally treated with NSF (100 or 200 mg/kg) or omeprazole (10 mg/kg) once a day along with indomethacin for five days. After sacrifice of the rats, gastric lesions were imaged using an optical digital camera. Formation of stomach lesions was evaluated using a pixel counter. (C) The pH of collected gastric juice was measured by a pH meter. (D, E) Histological examination of sections of gastric tissue stained with hematoxylin and eosin. Images were captured using an optical digital camera. Thicknesses of gastric walls were measured using ImageJ. (F) COX-1 mRNA level was determined by real-time PCR. (G) Protein levels of COX-1 and β-actin were determined by immunoblotting. (H) MPO activity as an indicator of neutrophil infiltration in the stomach was analyzed in total lysates of stomach. # p

    Article Snippet: COX-1 and β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Staining, Real-time Polymerase Chain Reaction, Activity Assay

    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

    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

    NanoATV and HIV-1 endosomal protein regulation. Western blot of Rab5, −7, −11, LAMP1 and β-actin was performed in cell lysates from MDM treated with native ATV or nanoATV and infected with HIV-1 at day 0, 5 or 10 post-drug treatment then incubated for 7 days. Uninfected cells and infected cells without drug treatment served as negative and positive controls for differential expression of cellular proteins during HIV-1 infection. Blots shown are from one donor and experiment, and equivalent to two independent experiments performed.

    Journal: Retrovirology

    Article Title: Opposing regulation of endolysosomal pathways by long-acting nanoformulated antiretroviral therapy and HIV-1 in human macrophages

    doi: 10.1186/s12977-014-0133-5

    Figure Lengend Snippet: NanoATV and HIV-1 endosomal protein regulation. Western blot of Rab5, −7, −11, LAMP1 and β-actin was performed in cell lysates from MDM treated with native ATV or nanoATV and infected with HIV-1 at day 0, 5 or 10 post-drug treatment then incubated for 7 days. Uninfected cells and infected cells without drug treatment served as negative and positive controls for differential expression of cellular proteins during HIV-1 infection. Blots shown are from one donor and experiment, and equivalent to two independent experiments performed.

    Article Snippet: Rabbit anti-human Rab 5, −7, −11, LAMP1 and β-actin antibodies were purchased from Santa Cruz Biotechnology, Dallas, TX, USA.

    Techniques: Western Blot, Infection, Incubation, Expressing

    BCL6 is required for the maintenance of LSK + cells in CML. (A and B) BCR-ABL1 transformed CML-like cells from BCL6 +/+ and BCL6 −/− bone marrow at 1 and 3 wk after transduction. Cells were gated on Lin − phenotype and surface expression of Sca-1 and c-Kit (LSK; A) and CD44 and c-kit (B) is shown ( n = 3). (C) Human CML cells (JURL cell line) were subjected to one round of ChIP-seq analysis for a genome-wide mapping analysis of recruitment of the BCL6 transcription factor. Overlays of input (green) and BCL6 ChIP (red) are shown for BCL6 (positive control; binding to its own promoter), HPRT (negative control), DNA damage response and cell cycle checkpoint genes including CHEK , CDKN2AIP , TP53 , CDKN1A (p21), GADD45A , and CDKN2A (Arf). Peaks of significant enrichment of BCL6 in promoter regions relative to input were identified by ChIPSeeqer (black bars). (D) BCR-ABL1–transformed CML-like cells from BCL6 +/+ and BCL6 −/− bone marrow were analyzed by Western blot for Arf and p53 protein levels using β-actin as loading control (two experiments are shown). (E) 100,000 BCL6 +/+ and BCL6 −/− CML-like cells were plated in semisolid methylcellulose agar and colonies were counted after 22 d. Chart shows mean values ± SD and p-value of 5 experiments. (F) Cell cycle analysis of BCL6 +/+ and BCL6 −/− CML cells was performed studying BrdU incorporation in combination with 7AAD staining. Annotations indicate distribution of CML-like cells to G0/1, S, and G2/M phases of the cell cycle. One representative experiment of three is shown.

    Journal: The Journal of Experimental Medicine

    Article Title: BCL6-mediated repression of p53 is critical for leukemia stem cell survival in chronic myeloid leukemia

    doi: 10.1084/jem.20110304

    Figure Lengend Snippet: BCL6 is required for the maintenance of LSK + cells in CML. (A and B) BCR-ABL1 transformed CML-like cells from BCL6 +/+ and BCL6 −/− bone marrow at 1 and 3 wk after transduction. Cells were gated on Lin − phenotype and surface expression of Sca-1 and c-Kit (LSK; A) and CD44 and c-kit (B) is shown ( n = 3). (C) Human CML cells (JURL cell line) were subjected to one round of ChIP-seq analysis for a genome-wide mapping analysis of recruitment of the BCL6 transcription factor. Overlays of input (green) and BCL6 ChIP (red) are shown for BCL6 (positive control; binding to its own promoter), HPRT (negative control), DNA damage response and cell cycle checkpoint genes including CHEK , CDKN2AIP , TP53 , CDKN1A (p21), GADD45A , and CDKN2A (Arf). Peaks of significant enrichment of BCL6 in promoter regions relative to input were identified by ChIPSeeqer (black bars). (D) BCR-ABL1–transformed CML-like cells from BCL6 +/+ and BCL6 −/− bone marrow were analyzed by Western blot for Arf and p53 protein levels using β-actin as loading control (two experiments are shown). (E) 100,000 BCL6 +/+ and BCL6 −/− CML-like cells were plated in semisolid methylcellulose agar and colonies were counted after 22 d. Chart shows mean values ± SD and p-value of 5 experiments. (F) Cell cycle analysis of BCL6 +/+ and BCL6 −/− CML cells was performed studying BrdU incorporation in combination with 7AAD staining. Annotations indicate distribution of CML-like cells to G0/1, S, and G2/M phases of the cell cycle. One representative experiment of three is shown.

    Article Snippet: Antibodies against β-actin were used as a loading control (H4; Santa Cruz Biotechnology, Inc.).

    Techniques: Transformation Assay, Transduction, Expressing, Chromatin Immunoprecipitation, Genome Wide, Positive Control, Binding Assay, Negative Control, Western Blot, Cell Cycle Assay, BrdU Incorporation Assay, Staining

    Regulation of BCL6 expression in CML cells. (A) To identify TKI-regulated genes in human CML cells, three human CML cell lines (KCL22, KU812, and JURL) were treated with (IM) or without (Ctrl) 1 μmol/liter Imatinib for 16 h and studied in an Affymetrix GeneChip analysis. Genes were sorted based on gene expression differences between TKI-treated and untreated CML cells. Likewise, BCR-ABL1 –transformed leukemia cells from Stat5 fl/fl bone marrow were transduced with Cre or an empty vector control. Gene expression values for Stat5 fl/fl (Ctrl) and Stat5-deleted (Cre) leukemia cells are indicated for the genes identified in TKI-treated cells. (B) Affymetrix GeneChip data for BCL6 was validated by quantitative RT-PCR on three cases of human CML (with or without 1 μmol/liter Imatinib overnight). (C) BCL6 gene expression values for CML cells from six patients before and after 7 d of Imatinib-treatment are shown (meta-analysis of data from Bruennert et al. [2009] ). (D) Human CML cells were treated with TKI (10 µM for 24 h) and BCL6 expression was evaluated by Western blotting using β-actin as a loading control. (E and F) Leukapheresis samples from two patients with CML-CP (CP21 and CP22) were sorted into four subpopulations based on CD34 and CD38 surface expression as depicted in the flow cytometry dot plots. Subpopulations were incubated overnight in the presence and absence of 10 µmol/liter Imatinib and then subjected to quantitative RT-PCR for BCL6 mRNA levels using COX6B as a reference gene. For each subpopulation, fold-induction of BCL6 mRNA levels are shown (triplicate measurements were performed; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: BCL6-mediated repression of p53 is critical for leukemia stem cell survival in chronic myeloid leukemia

    doi: 10.1084/jem.20110304

    Figure Lengend Snippet: Regulation of BCL6 expression in CML cells. (A) To identify TKI-regulated genes in human CML cells, three human CML cell lines (KCL22, KU812, and JURL) were treated with (IM) or without (Ctrl) 1 μmol/liter Imatinib for 16 h and studied in an Affymetrix GeneChip analysis. Genes were sorted based on gene expression differences between TKI-treated and untreated CML cells. Likewise, BCR-ABL1 –transformed leukemia cells from Stat5 fl/fl bone marrow were transduced with Cre or an empty vector control. Gene expression values for Stat5 fl/fl (Ctrl) and Stat5-deleted (Cre) leukemia cells are indicated for the genes identified in TKI-treated cells. (B) Affymetrix GeneChip data for BCL6 was validated by quantitative RT-PCR on three cases of human CML (with or without 1 μmol/liter Imatinib overnight). (C) BCL6 gene expression values for CML cells from six patients before and after 7 d of Imatinib-treatment are shown (meta-analysis of data from Bruennert et al. [2009] ). (D) Human CML cells were treated with TKI (10 µM for 24 h) and BCL6 expression was evaluated by Western blotting using β-actin as a loading control. (E and F) Leukapheresis samples from two patients with CML-CP (CP21 and CP22) were sorted into four subpopulations based on CD34 and CD38 surface expression as depicted in the flow cytometry dot plots. Subpopulations were incubated overnight in the presence and absence of 10 µmol/liter Imatinib and then subjected to quantitative RT-PCR for BCL6 mRNA levels using COX6B as a reference gene. For each subpopulation, fold-induction of BCL6 mRNA levels are shown (triplicate measurements were performed; *, P

    Article Snippet: Antibodies against β-actin were used as a loading control (H4; Santa Cruz Biotechnology, Inc.).

    Techniques: Expressing, Transformation Assay, Transduction, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Flow Cytometry, Cytometry, Incubation

    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

    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

    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

    Blimp1 promotes lung cancer cell migration and is aberrantly expressed in multiple cancers. (A) A549 cells or (B) H441 cells were transiently transfected with 1 µg of Blimp1 cDNA or EV DNA using Lipofectamine 2000. Upper panels: WCE were isolated after 48 h and subjected to immunoblot analysis for Blimp1 and β-actin. Lower panels: Alternatively, 24 h after transfection, cells were subjected to a migration assay as in Fig. 1 . The average migration from three independent experiments ± SD is presented relative to the EV (set at 1.0). P values were calculated using a Student's t -test. *, P

    Journal: PLoS ONE

    Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

    doi: 10.1371/journal.pone.0033287

    Figure Lengend Snippet: Blimp1 promotes lung cancer cell migration and is aberrantly expressed in multiple cancers. (A) A549 cells or (B) H441 cells were transiently transfected with 1 µg of Blimp1 cDNA or EV DNA using Lipofectamine 2000. Upper panels: WCE were isolated after 48 h and subjected to immunoblot analysis for Blimp1 and β-actin. Lower panels: Alternatively, 24 h after transfection, cells were subjected to a migration assay as in Fig. 1 . The average migration from three independent experiments ± SD is presented relative to the EV (set at 1.0). P values were calculated using a Student's t -test. *, P

    Article Snippet: Antibodies against β-actin (AC-15) and α-tubulin (DM1A) were from Sigma.

    Techniques: Migration, Transfection, Isolation

    A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 µg of a plasmid expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 µg) of WCE were subjected to immunoblot analysis for Blimp1, Ras and α-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH . The values represent an average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 µg of Ras S186 plasmid or EV DNA, 0.33 µg of a MSV- β-gal expression vector and 0.16 µg of the 7-kB Blimp1 promoter Blimp1 -Luc, in a 12-well plate. After 48 h, cell lysates were subjected to measurements for luciferase and β-gal activities and normalized Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and α-tubulin. Average normalized levels of Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c- RAF or a negative control siRNA was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and α-tubulin. Average normalized levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1 -luc promoter construct (0.16 µg) and an MSV- β-gal expression vector (0.33 µg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

    Journal: PLoS ONE

    Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

    doi: 10.1371/journal.pone.0033287

    Figure Lengend Snippet: A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 µg of a plasmid expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 µg) of WCE were subjected to immunoblot analysis for Blimp1, Ras and α-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH . The values represent an average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 µg of Ras S186 plasmid or EV DNA, 0.33 µg of a MSV- β-gal expression vector and 0.16 µg of the 7-kB Blimp1 promoter Blimp1 -Luc, in a 12-well plate. After 48 h, cell lysates were subjected to measurements for luciferase and β-gal activities and normalized Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and α-tubulin. Average normalized levels of Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c- RAF or a negative control siRNA was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and α-tubulin. Average normalized levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1 -luc promoter construct (0.16 µg) and an MSV- β-gal expression vector (0.33 µg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

    Article Snippet: Antibodies against β-actin (AC-15) and α-tubulin (DM1A) were from Sigma.

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Dominant Negative Mutation, Software, Isolation, Polymerase Chain Reaction, Luciferase, Negative Control, Incubation, Concentration Assay, Western Blot, Construct

    Blimp1 is expressed in lung cancer cells and its knockdown reduces migration. (A) Samples of nuclear extracts (20 µg) of A549, H1299, Calu-1, H23 and H441 human lung cancer cells and MCF-7 and MDA-MB-231 (MB-231) breast cancer cells were subjected to immunoblotting for Blimp1 and β-actin, as a control for equal loading. Positions of molecular weight markers are given in the left lane. A representative of two independent experiments with similar results is shown. (B) A549 and (C) H1299 cells were transiently transfected with 10 nM each of siBLIMP1-1 , siBLIMP1-2 or a scrambled negative control siRNA. Upper panels: Forty-eight h after transfection, WCE (30 µg) were subjected to immunoblotting for Blimp1 and β-actin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. Normalized Blimp1 expression was determined in two independent experiments and the average values are given below the blots. Lower panels: Alternatively, after 24 h, cultures were trypsinized and 1×10 5 cells subjected to a migration assay for 16 h, in triplicate. The average migration from three independent experiments ± SD is presented relative to the negative control siRNA (set at 1.0). P values were calculated using Student's t -test. *, P

    Journal: PLoS ONE

    Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

    doi: 10.1371/journal.pone.0033287

    Figure Lengend Snippet: Blimp1 is expressed in lung cancer cells and its knockdown reduces migration. (A) Samples of nuclear extracts (20 µg) of A549, H1299, Calu-1, H23 and H441 human lung cancer cells and MCF-7 and MDA-MB-231 (MB-231) breast cancer cells were subjected to immunoblotting for Blimp1 and β-actin, as a control for equal loading. Positions of molecular weight markers are given in the left lane. A representative of two independent experiments with similar results is shown. (B) A549 and (C) H1299 cells were transiently transfected with 10 nM each of siBLIMP1-1 , siBLIMP1-2 or a scrambled negative control siRNA. Upper panels: Forty-eight h after transfection, WCE (30 µg) were subjected to immunoblotting for Blimp1 and β-actin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. Normalized Blimp1 expression was determined in two independent experiments and the average values are given below the blots. Lower panels: Alternatively, after 24 h, cultures were trypsinized and 1×10 5 cells subjected to a migration assay for 16 h, in triplicate. The average migration from three independent experiments ± SD is presented relative to the negative control siRNA (set at 1.0). P values were calculated using Student's t -test. *, P

    Article Snippet: Antibodies against β-actin (AC-15) and α-tubulin (DM1A) were from Sigma.

    Techniques: Migration, Multiple Displacement Amplification, Molecular Weight, Transfection, Negative Control, Software, Expressing

    Ectopic LOX-PP reduces Blimp1 expression in lung cancer cells. (A) H1299-EV cells, and H1299-LOX-PP4 (PP4) and H1299-LOX-PP7 (PP7) clones, isolated as described previously [25] , were treated in triplicate with 2 µg/ml dox for 48 h. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). (B) A549-EV, A549-hLOX-PP, A549-mLOX-PP dox-inducible stable populations were treated with 2 µg/ml dox for 48 h in DMEM supplemented with 0.5% FBS. FBS was added back to 10% and cells incubated overnight. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). Samples of medium (5 ml) were subjected to immunoprecipitation followed by immunoblotting using V5 antibody for LOX-PP expression. (C) A549 and H1299 cells were transiently transfected with human LOX-PP cDNA or EV DNA. After 48 h, media and WCE were prepared. Samples of media (50 µl) were subjected to immunoblotting for V5. Samples of WCE (25 µg) were probed for Blimp1 and β-actin, and average normalized Blimp1 values from two independent experiments presented relative to EV DNA, set to 1.0. (D) A549 and H441 cells were treated with purified recombinant LOX-PP protein at a final concentration of 4 or 1 µg/ml, respectively, or the same volume of vehicle (water) in medium with 0.5% FBS. Twenty-four h later, FBS was added back to 10% and cultures incubated overnight. WCE were subjected to immunoblotting for Blimp1, phospho-c-Jun (p-c-Jun), total c-Jun, Fra-1 and Fra-2 and α-tubulin, as a loading control. Normalized Blimp1 and AP-1 subunit values from two independent experiments are presented relative to EV DNA, set to 1.0.

    Journal: PLoS ONE

    Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

    doi: 10.1371/journal.pone.0033287

    Figure Lengend Snippet: Ectopic LOX-PP reduces Blimp1 expression in lung cancer cells. (A) H1299-EV cells, and H1299-LOX-PP4 (PP4) and H1299-LOX-PP7 (PP7) clones, isolated as described previously [25] , were treated in triplicate with 2 µg/ml dox for 48 h. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). (B) A549-EV, A549-hLOX-PP, A549-mLOX-PP dox-inducible stable populations were treated with 2 µg/ml dox for 48 h in DMEM supplemented with 0.5% FBS. FBS was added back to 10% and cells incubated overnight. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). Samples of medium (5 ml) were subjected to immunoprecipitation followed by immunoblotting using V5 antibody for LOX-PP expression. (C) A549 and H1299 cells were transiently transfected with human LOX-PP cDNA or EV DNA. After 48 h, media and WCE were prepared. Samples of media (50 µl) were subjected to immunoblotting for V5. Samples of WCE (25 µg) were probed for Blimp1 and β-actin, and average normalized Blimp1 values from two independent experiments presented relative to EV DNA, set to 1.0. (D) A549 and H441 cells were treated with purified recombinant LOX-PP protein at a final concentration of 4 or 1 µg/ml, respectively, or the same volume of vehicle (water) in medium with 0.5% FBS. Twenty-four h later, FBS was added back to 10% and cultures incubated overnight. WCE were subjected to immunoblotting for Blimp1, phospho-c-Jun (p-c-Jun), total c-Jun, Fra-1 and Fra-2 and α-tubulin, as a loading control. Normalized Blimp1 and AP-1 subunit values from two independent experiments are presented relative to EV DNA, set to 1.0.

    Article Snippet: Antibodies against β-actin (AC-15) and α-tubulin (DM1A) were from Sigma.

    Techniques: Expressing, Isolation, Polymerase Chain Reaction, Incubation, Immunoprecipitation, Transfection, Purification, Recombinant, Concentration Assay

    Ectopic AP-1 subunits induce Blimp1 expression. (A) H441 cells, growing in 6-well plates, were transfected with 1 µg of vectors expressing the indicated AP-1 subunits or EV DNA (see bottom) to make a 2 µg total. Upper panel. After 48 h, RNA was isolated and subjected to Q-PCR. The levels of BLIMP1 mRNA normalized to GAPDH mRNA are presented as mean ± SD of three independent experiments. Middle and lower panels. WCE were isolated and subjected to immunoblotting (IB) for Blimp1 (Middle panels), and for c-Jun, Fra-1, Fra-2, c-Fos and β-actin (Lower panels). (L exp., longer exposure; S exp., shorter exposure). Blimp1 levels, normalized to β-actin, were determined as in Fig. 1C and average values from two independent experiments presented relative to EV DNA, set to 1.0. (B) H441 cells were transiently transfected, in triplicate, with 0.3 µg of Blimp1 -Luc, 0.3 µg of MSV-β-gal, and vectors expressing the indicated AP-1 subunits (0.15 µg each) and EV DNA to a total of 1.0 µg DNA. Normalized values of Blimp1 promoter activity are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

    Journal: PLoS ONE

    Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

    doi: 10.1371/journal.pone.0033287

    Figure Lengend Snippet: Ectopic AP-1 subunits induce Blimp1 expression. (A) H441 cells, growing in 6-well plates, were transfected with 1 µg of vectors expressing the indicated AP-1 subunits or EV DNA (see bottom) to make a 2 µg total. Upper panel. After 48 h, RNA was isolated and subjected to Q-PCR. The levels of BLIMP1 mRNA normalized to GAPDH mRNA are presented as mean ± SD of three independent experiments. Middle and lower panels. WCE were isolated and subjected to immunoblotting (IB) for Blimp1 (Middle panels), and for c-Jun, Fra-1, Fra-2, c-Fos and β-actin (Lower panels). (L exp., longer exposure; S exp., shorter exposure). Blimp1 levels, normalized to β-actin, were determined as in Fig. 1C and average values from two independent experiments presented relative to EV DNA, set to 1.0. (B) H441 cells were transiently transfected, in triplicate, with 0.3 µg of Blimp1 -Luc, 0.3 µg of MSV-β-gal, and vectors expressing the indicated AP-1 subunits (0.15 µg each) and EV DNA to a total of 1.0 µg DNA. Normalized values of Blimp1 promoter activity are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

    Article Snippet: Antibodies against β-actin (AC-15) and α-tubulin (DM1A) were from Sigma.

    Techniques: Expressing, Transfection, Isolation, Polymerase Chain Reaction, Activity Assay

    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

    Antibody targeting of sAPP-α promotes amyloidogenic APP processing in vivo . PSAPP mice (3 female mice per group) at 8 months of age were intracerebroventricular (i.c.v.) injected with 2B3 antibody or isotype-matched IgG 2b control antibody at 5 μg/mL and sacrificed at 48 h after the injection. ( a ) Mouse brain homogenates were prepared from these mice and subjected to IB analysis for APP processing. IB using 6E10 antibody shows total APP and three bands corresponding to β-CTF and soluble Aβ species 38 and 40. Densitometry analysis shows the ratios of Aβ 40 to β-actin ( b ) and β-CTF to β-actin ( c ). A t test revealed significant differences in either ratio of Aβ 40 to β-actin or β-CTF to β-actin between 2B3 antibody- and control IgG 2b antibody-i.c.v. injected PSAPP mice. (*** P

    Journal: Nature communications

    Article Title: sAPP-? modulates ?-secretase activity and amyloid-? generation

    doi: 10.1038/ncomms1781

    Figure Lengend Snippet: Antibody targeting of sAPP-α promotes amyloidogenic APP processing in vivo . PSAPP mice (3 female mice per group) at 8 months of age were intracerebroventricular (i.c.v.) injected with 2B3 antibody or isotype-matched IgG 2b control antibody at 5 μg/mL and sacrificed at 48 h after the injection. ( a ) Mouse brain homogenates were prepared from these mice and subjected to IB analysis for APP processing. IB using 6E10 antibody shows total APP and three bands corresponding to β-CTF and soluble Aβ species 38 and 40. Densitometry analysis shows the ratios of Aβ 40 to β-actin ( b ) and β-CTF to β-actin ( c ). A t test revealed significant differences in either ratio of Aβ 40 to β-actin or β-CTF to β-actin between 2B3 antibody- and control IgG 2b antibody-i.c.v. injected PSAPP mice. (*** P

    Article Snippet: Other antibodies include: mouse monoclonal BACE1 antibody (1 mg/mL; Millipore, Billerica, MA); rabbit polyclonal BACE1 antibody and APP C-terminal antibody (500 μg/mL; EMD Biosciences, La Jolla, CA); APP N-terminus antibody (100 μg/mL, Millopore); Aβ17-24 antibody (1 mg/mL; 4G8, Covance) and Aβ1-12 antibody (BAM10, 500μg/mL; Sigma-Aldrich, St. Louis, MO); β-actin antibody (100 μg/mL; Sigma-Aldrich); rabbit anti-APP C-terminus polyclonal antibody (500 μg/mL; pAb369) generously provided by Dr. Sam Gandy; rabbit anti-APP C-terminus polyclonal antibody (pAb751/770, 100 μg/mL; Calbiochem); rabbit polyclonal oligomer/conformational (OC) antibody (500 μg/mL) was provided by Dr. Suhail Rasool and Dr. Charles G. Glabe at University of California.

    Techniques: In Vivo, Mouse Assay, Injection

    Immunodepleted sAPP-α attenuates sAPP-α effects on APP modulation in vitro. Active hsAPP-α protein at 1 nM was incubated with an anti-C-terminal sAPP-α specific 2B3 antibody or isotype-matched IgG 2b control antibody at 2.5 and 5 μ/mL at 37°C for 30 min. The protein-G sepharose beads were used to pull-down sAPP-α antibody and isotype-matched IgG 2b control, as well as, their associated proteins. This was followed by high-speed centrifugation at 15,000 g for 10 min, and the supernatants were collected and determined to be IgG antibody free by IB analysis using an antibody against IgG 2b . These supernatants were used to treat CHO cells expressing human wild-type APP (CHO/APP wt ) for 12 h. Cell cultured supernatants were collected and subjected to ( a ) Aβ IB analysis using 6E10 antibody and ( b ) the Aβ ELISA results are represented as the mean ± SD of picograms of Aβ 40 or Aβ 42 per milligram of total intracellular protein after the 2B3- or control IgG 2b -immunodepleted hsAPP-α protein treatment. ( c ) Cell lysates were prepared and APP CTFs were analyzed by IB using pAb751/770 (C-APP). ( d ) Relative ratio (mean ± SD) of β-CTF to β-actin was calculated by densitometry analysis. For b and d , the results as presented are representative of two independent experiments with n = 3 for each condition. One-way ANOVA followed by post hoc comparison revealed significant differences between 2B3-immunodepleted hsAPP-α protein treatment at either 2.5 or 5 μg/mL and control IgG 2b -immunodepleted hsAPP-α protein as measured by either Aβ levels or ratio of β-CTF to β-actin. (*** P

    Journal: Nature communications

    Article Title: sAPP-? modulates ?-secretase activity and amyloid-? generation

    doi: 10.1038/ncomms1781

    Figure Lengend Snippet: Immunodepleted sAPP-α attenuates sAPP-α effects on APP modulation in vitro. Active hsAPP-α protein at 1 nM was incubated with an anti-C-terminal sAPP-α specific 2B3 antibody or isotype-matched IgG 2b control antibody at 2.5 and 5 μ/mL at 37°C for 30 min. The protein-G sepharose beads were used to pull-down sAPP-α antibody and isotype-matched IgG 2b control, as well as, their associated proteins. This was followed by high-speed centrifugation at 15,000 g for 10 min, and the supernatants were collected and determined to be IgG antibody free by IB analysis using an antibody against IgG 2b . These supernatants were used to treat CHO cells expressing human wild-type APP (CHO/APP wt ) for 12 h. Cell cultured supernatants were collected and subjected to ( a ) Aβ IB analysis using 6E10 antibody and ( b ) the Aβ ELISA results are represented as the mean ± SD of picograms of Aβ 40 or Aβ 42 per milligram of total intracellular protein after the 2B3- or control IgG 2b -immunodepleted hsAPP-α protein treatment. ( c ) Cell lysates were prepared and APP CTFs were analyzed by IB using pAb751/770 (C-APP). ( d ) Relative ratio (mean ± SD) of β-CTF to β-actin was calculated by densitometry analysis. For b and d , the results as presented are representative of two independent experiments with n = 3 for each condition. One-way ANOVA followed by post hoc comparison revealed significant differences between 2B3-immunodepleted hsAPP-α protein treatment at either 2.5 or 5 μg/mL and control IgG 2b -immunodepleted hsAPP-α protein as measured by either Aβ levels or ratio of β-CTF to β-actin. (*** P

    Article Snippet: Other antibodies include: mouse monoclonal BACE1 antibody (1 mg/mL; Millipore, Billerica, MA); rabbit polyclonal BACE1 antibody and APP C-terminal antibody (500 μg/mL; EMD Biosciences, La Jolla, CA); APP N-terminus antibody (100 μg/mL, Millopore); Aβ17-24 antibody (1 mg/mL; 4G8, Covance) and Aβ1-12 antibody (BAM10, 500μg/mL; Sigma-Aldrich, St. Louis, MO); β-actin antibody (100 μg/mL; Sigma-Aldrich); rabbit anti-APP C-terminus polyclonal antibody (500 μg/mL; pAb369) generously provided by Dr. Sam Gandy; rabbit anti-APP C-terminus polyclonal antibody (pAb751/770, 100 μg/mL; Calbiochem); rabbit polyclonal oligomer/conformational (OC) antibody (500 μg/mL) was provided by Dr. Suhail Rasool and Dr. Charles G. Glabe at University of California.

    Techniques: In Vitro, Incubation, Centrifugation, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    Immunoneutralization of endogenous sAPP-α promotes APP amyloidogenic processing. CHO/APP wt cells were treated with 2B3 or isotype-matched IgG 2b control antibody for 12 h as indicated. ( a ) Aβ species were analyzed in conditioned media from the treated CHO/APP wt cells by IB analysis using 6E10 antibody. ( b ) The Aβ ELISA results are represented as the mean ± SD of picograms of Aβ 40 or Aβ 42 per milligram of total intracellular protein after the 2B3 antibody treatment. In addition, these results are representative of three independent experiments with n = 3 for each condition. ( c ) Cell lysates were prepared and APP CTFs were analyzed by IB analysis using pAb751/770 (C-APP). ( d ) Densitometry shows the ratio (mean ± SD) of β-CTF to β-actin. For b , d , the results as presented are representative of three independent experiments with n = 3 for each condition. One-way ANOVA followed by post hoc comparison revealed a significant difference between the 2B3 antibody at either 2.5 or 5 μg/mL and isotype-matched IgG 2b control antibody treatment conditions. (*** P

    Journal: Nature communications

    Article Title: sAPP-? modulates ?-secretase activity and amyloid-? generation

    doi: 10.1038/ncomms1781

    Figure Lengend Snippet: Immunoneutralization of endogenous sAPP-α promotes APP amyloidogenic processing. CHO/APP wt cells were treated with 2B3 or isotype-matched IgG 2b control antibody for 12 h as indicated. ( a ) Aβ species were analyzed in conditioned media from the treated CHO/APP wt cells by IB analysis using 6E10 antibody. ( b ) The Aβ ELISA results are represented as the mean ± SD of picograms of Aβ 40 or Aβ 42 per milligram of total intracellular protein after the 2B3 antibody treatment. In addition, these results are representative of three independent experiments with n = 3 for each condition. ( c ) Cell lysates were prepared and APP CTFs were analyzed by IB analysis using pAb751/770 (C-APP). ( d ) Densitometry shows the ratio (mean ± SD) of β-CTF to β-actin. For b , d , the results as presented are representative of three independent experiments with n = 3 for each condition. One-way ANOVA followed by post hoc comparison revealed a significant difference between the 2B3 antibody at either 2.5 or 5 μg/mL and isotype-matched IgG 2b control antibody treatment conditions. (*** P

    Article Snippet: Other antibodies include: mouse monoclonal BACE1 antibody (1 mg/mL; Millipore, Billerica, MA); rabbit polyclonal BACE1 antibody and APP C-terminal antibody (500 μg/mL; EMD Biosciences, La Jolla, CA); APP N-terminus antibody (100 μg/mL, Millopore); Aβ17-24 antibody (1 mg/mL; 4G8, Covance) and Aβ1-12 antibody (BAM10, 500μg/mL; Sigma-Aldrich, St. Louis, MO); β-actin antibody (100 μg/mL; Sigma-Aldrich); rabbit anti-APP C-terminus polyclonal antibody (500 μg/mL; pAb369) generously provided by Dr. Sam Gandy; rabbit anti-APP C-terminus polyclonal antibody (pAb751/770, 100 μg/mL; Calbiochem); rabbit polyclonal oligomer/conformational (OC) antibody (500 μg/mL) was provided by Dr. Suhail Rasool and Dr. Charles G. Glabe at University of California.

    Techniques: Enzyme-linked Immunosorbent Assay

    sAPP-α inhibitsβ-secretase cleavage of APP in vitro . CHO cells expressing APP swe and wild-type human PS1 (CHO/APP swe /PS1 wt ) cells were treated with active (act.) or inactive (inact.) hsAPP-α recombinant protein at 0, 1, and 2 nM as indicated for 4 h. Secreted Aβ 40, 42 peptides in the cell culture medium were analyzed by ( a ) immunoblot (IB) and ( b ) Aβ ELISA analyses. The Aβ ELISA results are represented as the mean ± SD of picograms of Aβ 40 or Aβ 42 per milligram of total intracellular protein after hsAPP-α protein treatment. In addition, these results are representative of four independent experiments with n = 3 for each condition. ( c ) Cell lysates were prepared and APP CTFs were analyzed by IB using a rabbit polyclonal antibody against C-terminal APP (pAb751/770, C-APP). This β-CTF band was further confirmed by the additional IB using 6E10 antibody against Aβ 1-17 peptide for sAPP-α. ( d ) Relative ratio (mean ± SD) of β-CTF to β-actin was calculated by densitometry analysis. The results are representative of three independent experiments with n = 3 for each condition. One-way ANOVA followed by post hoc comparison revealed significant differences between 1 or 2 and 0 nM hsAPP-α protein treatment in both of Aβ 40, 42 reduction and relative ratio of β-CTF to β-actin. (*** P

    Journal: Nature communications

    Article Title: sAPP-? modulates ?-secretase activity and amyloid-? generation

    doi: 10.1038/ncomms1781

    Figure Lengend Snippet: sAPP-α inhibitsβ-secretase cleavage of APP in vitro . CHO cells expressing APP swe and wild-type human PS1 (CHO/APP swe /PS1 wt ) cells were treated with active (act.) or inactive (inact.) hsAPP-α recombinant protein at 0, 1, and 2 nM as indicated for 4 h. Secreted Aβ 40, 42 peptides in the cell culture medium were analyzed by ( a ) immunoblot (IB) and ( b ) Aβ ELISA analyses. The Aβ ELISA results are represented as the mean ± SD of picograms of Aβ 40 or Aβ 42 per milligram of total intracellular protein after hsAPP-α protein treatment. In addition, these results are representative of four independent experiments with n = 3 for each condition. ( c ) Cell lysates were prepared and APP CTFs were analyzed by IB using a rabbit polyclonal antibody against C-terminal APP (pAb751/770, C-APP). This β-CTF band was further confirmed by the additional IB using 6E10 antibody against Aβ 1-17 peptide for sAPP-α. ( d ) Relative ratio (mean ± SD) of β-CTF to β-actin was calculated by densitometry analysis. The results are representative of three independent experiments with n = 3 for each condition. One-way ANOVA followed by post hoc comparison revealed significant differences between 1 or 2 and 0 nM hsAPP-α protein treatment in both of Aβ 40, 42 reduction and relative ratio of β-CTF to β-actin. (*** P

    Article Snippet: Other antibodies include: mouse monoclonal BACE1 antibody (1 mg/mL; Millipore, Billerica, MA); rabbit polyclonal BACE1 antibody and APP C-terminal antibody (500 μg/mL; EMD Biosciences, La Jolla, CA); APP N-terminus antibody (100 μg/mL, Millopore); Aβ17-24 antibody (1 mg/mL; 4G8, Covance) and Aβ1-12 antibody (BAM10, 500μg/mL; Sigma-Aldrich, St. Louis, MO); β-actin antibody (100 μg/mL; Sigma-Aldrich); rabbit anti-APP C-terminus polyclonal antibody (500 μg/mL; pAb369) generously provided by Dr. Sam Gandy; rabbit anti-APP C-terminus polyclonal antibody (pAb751/770, 100 μg/mL; Calbiochem); rabbit polyclonal oligomer/conformational (OC) antibody (500 μg/mL) was provided by Dr. Suhail Rasool and Dr. Charles G. Glabe at University of California.

    Techniques: In Vitro, Expressing, Activated Clotting Time Assay, Recombinant, Cell Culture, Enzyme-linked Immunosorbent 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

    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

    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