anti-β-actin Search Results


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  • 96
    BioLegend anti β actin
    EGFR and PTK2 co-inhibition induce apoptosis in PC-9/PEM clone1. a Representative immunoblots of phosphorylated and total PTK2 in PC-9 and PC-9/PEM clone1 cells and the ratio of phosphorylated PTK2 to <t>β-actin.</t> Data are means (SD), n = 3. *, P
    Anti β Actin, supplied by BioLegend, used in various techniques. Bioz Stars score: 96/100, based on 287 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend purified anti β actin
    Dectin-1 expression in corneal epithelial tissue and primary cultured HCECs. Representative images showed immunofluorescent staining of dectin-1 on human corneal (A) and limbal tissue (B), as well as in HCECs (C). D. Dose-dependent stimulation of dectin-1 mRNA in HCECs by HKCA for 4 hours. E. Total protein of HCECs treated for 48 hours was extracted with RIPA buffer for western blot with dectin-1 or <t>β-actin</t> antibody. F. Quantitative ratio of the dectin-1/β-actin protein, evaluated by western blotting, in HCECs with or without exposure to 10 6 cell/ml of HKCA. Propidium iodide (PI) was used as nuclear counterstaining (red color). Magnification: 400Х (bar = 25μm). Data are presented as mean ± SD, n = 5; * p
    Purified Anti β Actin, supplied by BioLegend, used in various techniques. Bioz Stars score: 94/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proteintech anti β actin
    Alterations in the apoptotic signal proteins Bax and Bcl-2. SH-SY5Y cells were pretreated as indicated in 6-well plates. Bax (A) and Bcl-2 (B) protein levels were detected by Western blot analysis. Western blotting images were captured on a Bio-Imaging System (DNR Lumi BIS) using an ECL system and were analyzed by Image-Pro Plus 6.0. Representative bands of Bax, Bcl-2, and <t>β-actin</t> are shown. All bands were quantified and normalized by β-actin. (C) The Bax/Bcl-2 ratio is shown on the graph to demonstrate apoptosis more objectively. Statistical data are expressed as the mean±SEM from six independent experiment groups. * P
    Anti β Actin, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 866 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche anti β actin
    Proteomic analysis of the PSD fraction of GluN2B −/− cortical neurons. A , PSDs were successfully purified from high-density rat cortical cultures (14–15 DIV). Western blot for markers of the presynaptic (Synaptophysin) and postsynaptic fractions (PSD-95, SynGAP, NMDAR receptor subunits GluN1 and GluN2A, and AMPAR subunit GluA1) validated PSD isolation of PSDs. Transferrin receptor (Transf R) and <t>β-actin</t> were used as controls. Hom, Homogenate; S1, non-nuclear fraction; P2 crude synaptosomes. Equal amounts (13 μg) of each fraction were applied. B , PSD-localized proteins (PSD-95, GluN1, SynGAP) are increased relative to the homogenate in the PSD fraction, whereas presynaptic synaptophysin (Synapto.) or the trans membrane receptor of transferrin are decreased compared with the P2 fraction, consistent with their absence or low expression, respectively, at the PSD. Actin levels did not change in any of the fractions analyzed. Data are presented as mean ± SEM from two independent experiments. C , Setup of the 8-plex iTRAQ experiments. Isolated PSDs from GluN2B +/+ , GluN2B +/− , and GluN2B −/− cortical neurons, 10 μg each, were digested with trypsin and samples (three wild-types, two heterozygous, and three knock-outs) were tagged with a set of 8-plex iTRAQ reagents. Peptides were pooled together and fractionated by 2D liquid chromatography and subject to tandem mass spectrometric analysis. After using peptides for protein identification, the individual contribution of each sample to each peptide can be measured by the intensity of the reporter ion peaks and used for relative quantification. Example spectra correspond to one of the GluN1 peptides identified in the PSD preparations. D , Chymotrypsin-like proteasome activity was measure in protein extracts from GluN2B +/+ and GluN2B −/− cortical neurons. Proteasome activity is significantly decreased ( p
    Anti β Actin, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Epitomics anti β actin
    APH1B is involved in modulating stemness in SiHa oncospheres. Notes: ( A ) Heat-map of four stem cell pathway-related genes, WNT5B, APC2, WNT9A, and APH1B. ( B ) Real-time RT-PCR detection of the expression of WNT5B, APC2, WNT9A, and APH1B mRNAs in si-E7 and si-NC SiHa oncospheres. ( C ) Western blot detection of APH1B protein in si-E7 and si-NC SiHa oncospheres. ( D ) Phase-contrast photomicrographs of SiHa oncospheres with APH1B inhibition or overexpression in low-adherence cultures for 7 days. ( E ) Western blot detection of SOX2 and OCT4 proteins in SiHa oncospheres with APH1B inhibition or overexpression. ( F and G ) Growth inhibition of SiHa oncospheres with APH1B inhibition or overexpression. Both were seeded in 96-well plates and treated with paclitaxel or cisplatin at different concentrations (0, 1, 2, 5, 10, 20, 40, 60, 80, 100 nM) for 48 hrs, and cell viability was determined by a modified MTT assay. OD values of each treated group were compared with controls at the same time point. ( H ) Representative photomicrographs of clonal expansion of single oncospheres from SiHa with APH1B inhibition or overexpression in low-adherence cultures over a 7-day period. The cluster of oncospheres after days 1, 3, 5, 7 of culture was measured. Western blot expression levels were normalized to those of <t>β-actin.</t> Error bars and mean with SD were from three independent experiments. * P
    Anti β Actin, supplied by Epitomics, used in various techniques. Bioz Stars score: 93/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim anti β actin
    APH1B is involved in modulating stemness in SiHa oncospheres. Notes: ( A ) Heat-map of four stem cell pathway-related genes, WNT5B, APC2, WNT9A, and APH1B. ( B ) Real-time RT-PCR detection of the expression of WNT5B, APC2, WNT9A, and APH1B mRNAs in si-E7 and si-NC SiHa oncospheres. ( C ) Western blot detection of APH1B protein in si-E7 and si-NC SiHa oncospheres. ( D ) Phase-contrast photomicrographs of SiHa oncospheres with APH1B inhibition or overexpression in low-adherence cultures for 7 days. ( E ) Western blot detection of SOX2 and OCT4 proteins in SiHa oncospheres with APH1B inhibition or overexpression. ( F and G ) Growth inhibition of SiHa oncospheres with APH1B inhibition or overexpression. Both were seeded in 96-well plates and treated with paclitaxel or cisplatin at different concentrations (0, 1, 2, 5, 10, 20, 40, 60, 80, 100 nM) for 48 hrs, and cell viability was determined by a modified MTT assay. OD values of each treated group were compared with controls at the same time point. ( H ) Representative photomicrographs of clonal expansion of single oncospheres from SiHa with APH1B inhibition or overexpression in low-adherence cultures over a 7-day period. The cluster of oncospheres after days 1, 3, 5, 7 of culture was measured. Western blot expression levels were normalized to those of <t>β-actin.</t> Error bars and mean with SD were from three independent experiments. * P
    Anti β Actin, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Valiant anti β actin
    PI3K–Akt–Foxo3a activation in Cbl-b–mediated regulation of Foxp3 + iT reg cells. (A) Phosphorylation of Smad proteins in naive CD4 + T cells from WT, Cbl-b KO, and Cbl-b C373A mice. Naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 in the presence of TGF-β for indicated time periods. Whole-cell lysates were immunoblotted with anti–phospho-(p)-Smad2, or anti–p-Smad3, and reprobed with anti-Smad2/3. Smad2, 60 kD; Smad3, 52 kD. Data are representative of three independent experiments. (B) Naive CD4 + T cells from WT and Cbl-b KO mice were stimulated with anti-CD3 and anti-CD28 together with TGF-β in the absence or presence of the PI3K inhibitor LY294002 (LY), the JNK inhibitor SP600125 (SP), the p38 inhibitor SB203580 (SB), or the calcineurin inhibitor cyclosporine A (Csp A). Foxp3 expression was assessed by FACS analysis after 5 d. The percentages of Foxp3-expressing cells are shown. Data are representative of three independent experiments. (C) Naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 for the indicated time periods. Whole-cell lysates were immunoblotted with anti–p-Akt, anti-Akt, anti–p-Erk, anti-Erk2, and <t>anti–β-actin.</t> Akt, 56 kD; Erk, 42/44 kD; β-actin, 45 kD. Data are representative of at least three independent experiments. (D) Regulation of Foxo3a phosphorylation by Cbl-b E3 ligase. Naive CD4 + T cells from WT and Cbl-b KO mice (top) or WT and Cbl-b C373A mice (bottom) were stimulated with anti-CD3 and anti-CD28 for indicated time periods. Whole-cell lysates were immunoblotted with anti–p-Foxo3a, anti-Foxo3a, and anti–β-actin. Foxo3a, 95 kD; β-actin, 45 kD. Data are representative of at least three independent experiments.
    Anti β Actin, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti β actin
    Effects of perivascular adipose tissue (PVAT) on endothelial nitric oxide synthase (eNOS) expression and phosphorylated residues. The aorta homogenates in the presence and absence of PVAT were used to measure eNOS protein expression (n = 26∼27) (A), eNOS phosphorylation on Thr 495  (n = 7∼8) (B) or Ser 1177  (n = 11) (C), and adenosine monophosphate-activated protein kinase (AMPK) expression (n = 5) (D). Blots are representative of the numbers of animals in parentheses. Densitometric mean values were normalized to  β -actin or eNOS protein levels. (−) PVAT, absence of PVAT; (+) PVAT, presence of PVAT.
    Anti β Actin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 361 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bioss anti β actin
    Hepatic overexpression of HNF4 α  in rats decreased cholesterol levels in both plasma and liver. Rats were intravenously injected with Ad-HNF4 α  or Ad-GFP ( n  = 6), then killed 7 days post-infection. (a) Liver extracts were used to determine protein expression by western blot analysis with antibodies against HNF4 α  or  β -actin. (b) Relative mRNA levels of hepatic HNF4 α  target genes, including G6p, Apoc3, Apob, and L-pk, were examined by qPCR. (c) Plasma cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were determined. (d) Liver cholesterol was  measured in lipid content of rat liver. Data are mean ± SEM, * P
    Anti β Actin, supplied by Bioss, used in various techniques. Bioz Stars score: 92/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PRO Scientific anti β actin
    Hepatic overexpression of HNF4 α  in rats decreased cholesterol levels in both plasma and liver. Rats were intravenously injected with Ad-HNF4 α  or Ad-GFP ( n  = 6), then killed 7 days post-infection. (a) Liver extracts were used to determine protein expression by western blot analysis with antibodies against HNF4 α  or  β -actin. (b) Relative mRNA levels of hepatic HNF4 α  target genes, including G6p, Apoc3, Apob, and L-pk, were examined by qPCR. (c) Plasma cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were determined. (d) Liver cholesterol was  measured in lipid content of rat liver. Data are mean ± SEM, * P
    Anti β Actin, supplied by PRO Scientific, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ABclonal anti β actin
    Hepatic overexpression of HNF4 α  in rats decreased cholesterol levels in both plasma and liver. Rats were intravenously injected with Ad-HNF4 α  or Ad-GFP ( n  = 6), then killed 7 days post-infection. (a) Liver extracts were used to determine protein expression by western blot analysis with antibodies against HNF4 α  or  β -actin. (b) Relative mRNA levels of hepatic HNF4 α  target genes, including G6p, Apoc3, Apob, and L-pk, were examined by qPCR. (c) Plasma cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were determined. (d) Liver cholesterol was  measured in lipid content of rat liver. Data are mean ± SEM, * P
    Anti β Actin, supplied by ABclonal, used in various techniques. Bioz Stars score: 93/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies anti β actin
    Hepatic overexpression of HNF4 α  in rats decreased cholesterol levels in both plasma and liver. Rats were intravenously injected with Ad-HNF4 α  or Ad-GFP ( n  = 6), then killed 7 days post-infection. (a) Liver extracts were used to determine protein expression by western blot analysis with antibodies against HNF4 α  or  β -actin. (b) Relative mRNA levels of hepatic HNF4 α  target genes, including G6p, Apoc3, Apob, and L-pk, were examined by qPCR. (c) Plasma cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were determined. (d) Liver cholesterol was  measured in lipid content of rat liver. Data are mean ± SEM, * P
    Anti β Actin, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti β actin
    Pulmonary muscarinic receptors function and expression in allergic AIRmin and AIRmax mice. AIRmin or AIRmax mice were sensitized and challenged with OVA as in  Figure 4 . Control group consisted of nonmanipulated animals. The experiments were performed 24 h after the last OVA challenge. Respiratory pattern of allergic AIRmin (a and c) or AIRmax (b and d) to inhaled MCh was evaluated in the presence or absence of gallamine. Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh. Area under the curve was obtained from Penh values (c and d). Gene (e and g) or protein (f and h) expression of the M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from OVA-sensitized AIRmin and AIRmax mice. The real-time PCR was carried out using  β -actin gene expression as internal control for normalization of M3R (e) and M2R (g) mRNA transcription levels. In Western blot analysis the density of M3R (f) and M2R (h) protein expression was normalized to actin expression in each sample. Data are expressed as mean ± SEM of four mice per group and are representative of two experiments. Western blots data were quantified by densitometry using the ImageJ software (NIH). Statistical analyses of Student's  t  test for (a), (b), (e), (f), (g), and (h). Statistical analyses of ANOVA following Tukey HSD for (c) and (d). * P
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    Abcam anti β actin
    DBT regulates expressions of Bcl2 and Bax. Cultured cortical neurons were co-treated with various concentration of DBT (DBT-L: 12.5 μg/mL; DBT-H: 100 μg/mL) and Aβ (20 μM) for 48 h. The protein expression levels of Bcl2 (~ 27 kDa) and Bax (~ 21 kDa) were detected by immunoblot analysis using specific antibodies, and <t>β-actin</t> (~ 42 kDa) served as an internal control. Quantification of target protein expression was calculated by a densitometer. Values were expressed as the percentage of change as compared to control reading, in Mean ± SEM, where n = 3. Statistically significant changes were classified as more significant (**) where p
    Anti β Actin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8076 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TransGen biotech co anti β actin
    SMARCAD1 enhances proliferation of PANC-1 cells. A-B. The efficiency of SMARCAD1 knockdown (A) or overexpression (B) in PANC-1 cells was detected by western blotting. <t>β-actin</t> was used as an internal control. C-D. CCK8 assay was performed to determine the proliferation of PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) at the indicated time points after plated. Cell viability was measured at 450nm. E-F. The effect of SMARCAD1 knockdown (E) or overexpression (F) on Colony-forming of PANC-1 cells was shown in the top panels. Number of foci was counted as shown in the bottom panels. All data were presented as mean ±SEM. *p
    Anti β Actin, supplied by TransGen biotech co, used in various techniques. Bioz Stars score: 91/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ABM Inc anti β actin
    Induction of apoptotic and autophagic cell death in TMZ-resistant glioma cells by VPA and TMZ combination. T98 and U138 cells were untreated or treated with VPA, TMZ, or their combination. (a) The expression of MGMT, Bcl-2, Bak, and caspase-3 was evaluated using western blotting. (b) Conversion of LC3-I to LC3-II was also determined by western blotting.  β -actin was used as a loading control. The results are representative of three independent experiments.
    Anti β Actin, supplied by ABM Inc, used in various techniques. Bioz Stars score: 92/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Zhongshan Golden Bridge Company anti β actin
    Low dose of apelin-36 attenuates cerebral I/R injury-induced caspase-3 activation in rats. (A) The MCA territory of sham treated rats, I/R model rats and I/R model rats treated with apelin-36 (labeled I/R-Apelin) were lysed in RIPA-DOC buffer. Cell lysates were resolved on 12% Tris–Glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cleaved caspase-3 was detected by cleaved caspase-3 antibody and <t>β-actin</t> served as an internal control was detected by β-actin antibody. (B) Quantification of cleaved caspase-3 expression. The ratio of cleaved caspase-3 to β-actin was further normalized to the sham treated rats. Values represent mean ± SEM. N = 5, * p
    Anti β Actin, supplied by Zhongshan Golden Bridge Company, used in various techniques. Bioz Stars score: 92/100, based on 224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime anti β actin
    Sch A induces Nrf2 activation in tissues. Protein expression of Nrf2, HO-1, and NQO-1 (A) was evaluated by Western blotting analyses. (B-D) Quantification of Nrf2, HO-1, and NQO-1 levels normalized to that of <t>β-actin.</t> All data are mean ± SD. (##P
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    GeneTex anti β actin
    Photoreceptor protein expression in GCAP1 −/− retina. A. Immunoblots of SDS polyacrylamide gels containing samples from WT and GCAP1 KO retinas probed with antibodies raised against GCAP2, RetGC1, RetGC2, rod α-transducin (Gtα1), PDE6, arrestin 1 (ARR), GRK1, RGS9, and <t>β-actin,</t> as indicated. B. Average (± SD) integrated chemiluminescence signal intensity in the band for the corresponding antigen in GCAP1 −/− retina relative to the WT for GCAP1 (n = 5), GCAP2 (n = 7), RetGC1 (n = 3), RetGC2 (n = 3), rod α-transducin (n = 3), PDE6 (n = 3), arrestin (n = 3), GRK1 (n = 3), RGS9 (n = 3), and β-actin (n = 3). When compared by one-way ANOVA with Bonferroni’s post hoc test (alpha = 0.01), there were significant differences found in GCAP1 (**) and GCAP2 (*) contents (P
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    Bioworld Antibodies anti β actin
    Effects of NCBAE on 2K1C-induced apoptosis. (a) The expression of Bcl-2 and cleaved caspase-3 protein was determined by western blotting. (b) Protein levels were measured by densitometry and presented as histograms. Bcl-2 and cleaved caspase-3 protein normalized to  β -actin.  ∗ P
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    Merck KGaA anti β actin
    (a) Western blotting for EGFR (170 kDa) and Beclin1 (60 kDa) in representative H-EGFR/L-Beclin1 (lanes 1 and 2), L-EGFR/H-Beclin1 (lanes 3–5), H-EGFR/H-Beclin1 (lanes 6–8), and L-EGFR/L-Beclin1 (lanes 9–11) GBs. (b) Up- or downregulation of either EGFR or Beclin1 is quantified by densitometric data analysis, which shows the relative expression of EGFR and Beclin1 after normalization to the  β -actin bands. Data are reported as means ± S.E. of three densitometric analyses of the same sample.
    Anti β Actin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 209 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenScript anti β actin
    Nitrogen or carbon starvation induces autophagy in C. neoformans . (A,B) Analysis of GFP-Atg8 processing by western blotting. H99 and atg Δ strains transformed with a plasmid expressing the EGFP-Atg8 fusion protein were collected after shifting to nitrogen starvation medium (SD-N) (A) or carbon starvation medium (SD-C) (B) at the indicated times. Total proteins were extracted and subjected to immunoblot analysis; <t>β-actin</t> was used as the loading control. (C) Localization of EGFP-Atg8 upon nutrient starvation. H99, atg1 Δ and atg13 Δ cells in log phase (starved; 0 h) or after shifting to SD-N or SD-C medium containing 10 μg/mL nocodazole for 4 h were observed by both fluorescence microscopy and differential interferential contrast (DIC) with a laser scanning confocal microscope. (D) Autophagic body formation. H99 and atg Δ strains were incubated in SD-N or SD-C medium containing 1 mM PMSF and 10 μg/mL nocodazole for 4 h and subjected to DIC microscopy. Arrows indicate autophagic bodies within vacuoles.
    Anti β Actin, supplied by GenScript, used in various techniques. Bioz Stars score: 90/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti β actin
    IL-5 and CD40L stimulation expands a population of B cells enriched for FasL expression. ( A ) Diagram of B cell co-culture experiments with CD40L-expressing fibroblasts for study of the effects of IL-5 on B cell growth and function. ( B ) CD19 + B cells were cultured as illustrated in (A) and harvested after five days. Viable B cells recovered from culture were quantified from replicate samples using a hemocytometer and trypan blue exclusion. The dotted line represents the concentration of B cells at the beginning of the experiment. ( C ) Proliferation of B cells cultured with CD40L-expressing fibroblasts in the presence or absence of IL-5 was assessed by the incorporation of 3 H-thymidine. ( D ) B cells stimulated with CD40L in the presence or absence of IL-5 were stained for FasL, CD5, and CD1d and compared with freshly-isolated CD19 + B cells. ( E ) Median fluorescence intensity of surface markers stained as in (D) on freshly-isolated CD19 + B cells, B cells stimulated with CD40L, and B cells stimulated with CD40L and IL-5. ( F ) The frequency of FasL + B cells among freshly-isolated CD19 + B cells and B cells stimulated with CD40L and IL-5 was measured by flow cytometry as in (D). ( G ) Cell lysates from equal numbers of freshly-isolated CD19 + B cells and IL-5-stimulated B cells were probed by immunoblot for FasL and <t>β-Actin.</t> Data are representative of more than four independent experiments. *** p
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    Image Search Results


    EGFR and PTK2 co-inhibition induce apoptosis in PC-9/PEM clone1. a Representative immunoblots of phosphorylated and total PTK2 in PC-9 and PC-9/PEM clone1 cells and the ratio of phosphorylated PTK2 to β-actin. Data are means (SD), n = 3. *, P

    Journal: Respiratory Research

    Article Title: Protein tyrosine kinase 2: a novel therapeutic target to overcome acquired EGFR-TKI resistance in non-small cell lung cancer

    doi: 10.1186/s12931-019-1244-2

    Figure Lengend Snippet: EGFR and PTK2 co-inhibition induce apoptosis in PC-9/PEM clone1. a Representative immunoblots of phosphorylated and total PTK2 in PC-9 and PC-9/PEM clone1 cells and the ratio of phosphorylated PTK2 to β-actin. Data are means (SD), n = 3. *, P

    Article Snippet: The following were primary antibodies: anti-EGFR, anti-pEGFR (Y1068), anti-PARP, anti-cPARP (D214), anti-pAkt (S473), anti-ERK1/2, anti-pERK1/2 (T202/Y204), anti-PTK2, anti-pPTK2 (Y566/567), anti-FGFR1 (D8E4), anti-pFGFR (Y653/654), anti-FGFR4 (D3B12) (Cell Signaling Technology, Danvers, MA, USA), anti-Akt (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-β-actin (BioLegend, San Diego, CA, USA).

    Techniques: Inhibition, End-sequence Profiling, Western Blot

    SOCS1 negatively regulates DCs in vitro . (A) DC2.4 cells were transfected with SOCS1 siRNAs (siRNA1-3) using Lipofectamine 2000. The protein expression levels of SOCS1, pSTAT1 and STAT1 in DC2.4 cells 48 h after transfection with SOCS1 siRNA were determined by western blot analysis. Representative western blot analysis results from one of three independent experiments are demonstrated. The intensity of (B) SOCS1 and (C) pSTAT1 bands were normalized to that of the β-actin bands, and the relative intensities (ratios) are presented. (D) Levels of IFN-γ secretion by siRNA-transfected or mock-transfected DC2.4 cells in response to 100 ng/ml LPS-simulation for 24 h. *P

    Journal: Oncology Letters

    Article Title: Silencing of suppressor of cytokine signaling 1 enhances the immunological effect of mucin 1-calreticulin-primed 4T1 cell-treated dendritic cells in breast cancer treatment

    doi: 10.3892/ol.2017.7477

    Figure Lengend Snippet: SOCS1 negatively regulates DCs in vitro . (A) DC2.4 cells were transfected with SOCS1 siRNAs (siRNA1-3) using Lipofectamine 2000. The protein expression levels of SOCS1, pSTAT1 and STAT1 in DC2.4 cells 48 h after transfection with SOCS1 siRNA were determined by western blot analysis. Representative western blot analysis results from one of three independent experiments are demonstrated. The intensity of (B) SOCS1 and (C) pSTAT1 bands were normalized to that of the β-actin bands, and the relative intensities (ratios) are presented. (D) Levels of IFN-γ secretion by siRNA-transfected or mock-transfected DC2.4 cells in response to 100 ng/ml LPS-simulation for 24 h. *P

    Article Snippet: Purified anti-β-actin antibody was purchased from Biolegend, Inc. (San Diego, CA, USA; cat. no. 643801).

    Techniques: In Vitro, Transfection, Expressing, Western Blot

    Dectin-1 expression in corneal epithelial tissue and primary cultured HCECs. Representative images showed immunofluorescent staining of dectin-1 on human corneal (A) and limbal tissue (B), as well as in HCECs (C). D. Dose-dependent stimulation of dectin-1 mRNA in HCECs by HKCA for 4 hours. E. Total protein of HCECs treated for 48 hours was extracted with RIPA buffer for western blot with dectin-1 or β-actin antibody. F. Quantitative ratio of the dectin-1/β-actin protein, evaluated by western blotting, in HCECs with or without exposure to 10 6 cell/ml of HKCA. Propidium iodide (PI) was used as nuclear counterstaining (red color). Magnification: 400Х (bar = 25μm). Data are presented as mean ± SD, n = 5; * p

    Journal: PLoS ONE

    Article Title: A Novel Innate Response of Human Corneal Epithelium to Heat-killed Candida albicans by Producing Peptidoglycan Recognition Proteins

    doi: 10.1371/journal.pone.0128039

    Figure Lengend Snippet: Dectin-1 expression in corneal epithelial tissue and primary cultured HCECs. Representative images showed immunofluorescent staining of dectin-1 on human corneal (A) and limbal tissue (B), as well as in HCECs (C). D. Dose-dependent stimulation of dectin-1 mRNA in HCECs by HKCA for 4 hours. E. Total protein of HCECs treated for 48 hours was extracted with RIPA buffer for western blot with dectin-1 or β-actin antibody. F. Quantitative ratio of the dectin-1/β-actin protein, evaluated by western blotting, in HCECs with or without exposure to 10 6 cell/ml of HKCA. Propidium iodide (PI) was used as nuclear counterstaining (red color). Magnification: 400Х (bar = 25μm). Data are presented as mean ± SD, n = 5; * p

    Article Snippet: The membranes were blocked with 5% nonfat milk in TTBS (50 mM Tris [pH 7.5], 0.9% NaCl, and 0.1% Tween-20) for 1 hour at room temperature and incubated with primary antibodies to dectin-1 (1:200), PGLYRP-2 (1:200), or β-actin (622102, BioLegend, 1:1000) overnight at 4°C.

    Techniques: Expressing, Cell Culture, Staining, Western Blot

    Dectin-1 and NF-κB signaling pathways involve PGLYRPs induction in HCECs exposed to HKCA. A. The HCECs were exposed to 10 6 cells/ml HKCA with prior incubation in the absence or presence of isotype IgG (10μg/ml), dectin-1 Ab (10μg/ml), BAY11-7082 (10μM) or NF-κB activation inhibitor quinazoline (NF-κB-I, 10μM) for 1 h. Cultures treated by HKCA for 4 h were subjected to RT-qPCR to measure mRNA. B. Total protein of HCECs treated for 48 hours was extracted with RIPA buffer for western blot to examine PGLYRP-2 production. C. Protein levels of PGLYRP-2 were evaluated by western blot using β-actin as control with quantitative ratio of PGLYRP-2/β-actin. Results shown are the mean ± SD of four independent experiments; *** p

    Journal: PLoS ONE

    Article Title: A Novel Innate Response of Human Corneal Epithelium to Heat-killed Candida albicans by Producing Peptidoglycan Recognition Proteins

    doi: 10.1371/journal.pone.0128039

    Figure Lengend Snippet: Dectin-1 and NF-κB signaling pathways involve PGLYRPs induction in HCECs exposed to HKCA. A. The HCECs were exposed to 10 6 cells/ml HKCA with prior incubation in the absence or presence of isotype IgG (10μg/ml), dectin-1 Ab (10μg/ml), BAY11-7082 (10μM) or NF-κB activation inhibitor quinazoline (NF-κB-I, 10μM) for 1 h. Cultures treated by HKCA for 4 h were subjected to RT-qPCR to measure mRNA. B. Total protein of HCECs treated for 48 hours was extracted with RIPA buffer for western blot to examine PGLYRP-2 production. C. Protein levels of PGLYRP-2 were evaluated by western blot using β-actin as control with quantitative ratio of PGLYRP-2/β-actin. Results shown are the mean ± SD of four independent experiments; *** p

    Article Snippet: The membranes were blocked with 5% nonfat milk in TTBS (50 mM Tris [pH 7.5], 0.9% NaCl, and 0.1% Tween-20) for 1 hour at room temperature and incubated with primary antibodies to dectin-1 (1:200), PGLYRP-2 (1:200), or β-actin (622102, BioLegend, 1:1000) overnight at 4°C.

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

    Alterations in the apoptotic signal proteins Bax and Bcl-2. SH-SY5Y cells were pretreated as indicated in 6-well plates. Bax (A) and Bcl-2 (B) protein levels were detected by Western blot analysis. Western blotting images were captured on a Bio-Imaging System (DNR Lumi BIS) using an ECL system and were analyzed by Image-Pro Plus 6.0. Representative bands of Bax, Bcl-2, and β-actin are shown. All bands were quantified and normalized by β-actin. (C) The Bax/Bcl-2 ratio is shown on the graph to demonstrate apoptosis more objectively. Statistical data are expressed as the mean±SEM from six independent experiment groups. * P

    Journal: Acta Pharmacologica Sinica

    Article Title: Inhibition of tissue transglutaminase promotes Aβ-induced apoptosis in SH-SY5Y cells

    doi: 10.1038/aps.2016.95

    Figure Lengend Snippet: Alterations in the apoptotic signal proteins Bax and Bcl-2. SH-SY5Y cells were pretreated as indicated in 6-well plates. Bax (A) and Bcl-2 (B) protein levels were detected by Western blot analysis. Western blotting images were captured on a Bio-Imaging System (DNR Lumi BIS) using an ECL system and were analyzed by Image-Pro Plus 6.0. Representative bands of Bax, Bcl-2, and β-actin are shown. All bands were quantified and normalized by β-actin. (C) The Bax/Bcl-2 ratio is shown on the graph to demonstrate apoptosis more objectively. Statistical data are expressed as the mean±SEM from six independent experiment groups. * P

    Article Snippet: Anti-Bax, anti-Bcl-2, anti-β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from ProteinTech (Chicago, IL, USA).

    Techniques: Western Blot, Imaging

    Western blotting analysis of tTG and isopeptide in NTU283- and Aβ-treated SH-SY5Y cells. Cells were seeded in 6-well plates. After 6 d of administration of RA, serum-free medium containing different concentrations of NTU283 (0, 20, 100, and 500 μmol/L) with or without 1 μmol/L Aβ was added to each well. Cells were harvested after a 24-h treatment. Western blot analysis was applied to detect tTG and isopeptide protein levels. Western blotting images were collected using a Bio-Imaging System (DNR Lumi BIS) with an ECL system and analyzed by Image-Pro Plus 6.0. (A) Representative Western blots for tTG protein levels (top) and quantitative analysis (bottom) are shown. (B) Representative Western blots for isopeptide protein levels (top) and quantitative analysis (bottom) are shown. All bands were quantified and normalized by β-actin. Statistical data are expressed as the mean±SEM from six independent experiment. * P

    Journal: Acta Pharmacologica Sinica

    Article Title: Inhibition of tissue transglutaminase promotes Aβ-induced apoptosis in SH-SY5Y cells

    doi: 10.1038/aps.2016.95

    Figure Lengend Snippet: Western blotting analysis of tTG and isopeptide in NTU283- and Aβ-treated SH-SY5Y cells. Cells were seeded in 6-well plates. After 6 d of administration of RA, serum-free medium containing different concentrations of NTU283 (0, 20, 100, and 500 μmol/L) with or without 1 μmol/L Aβ was added to each well. Cells were harvested after a 24-h treatment. Western blot analysis was applied to detect tTG and isopeptide protein levels. Western blotting images were collected using a Bio-Imaging System (DNR Lumi BIS) with an ECL system and analyzed by Image-Pro Plus 6.0. (A) Representative Western blots for tTG protein levels (top) and quantitative analysis (bottom) are shown. (B) Representative Western blots for isopeptide protein levels (top) and quantitative analysis (bottom) are shown. All bands were quantified and normalized by β-actin. Statistical data are expressed as the mean±SEM from six independent experiment. * P

    Article Snippet: Anti-Bax, anti-Bcl-2, anti-β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from ProteinTech (Chicago, IL, USA).

    Techniques: Western Blot, Imaging

    In-cell Western analysis of tTG and isopeptide in NTU283- and Aβ-treated SH-SY5Y cells. SH-SY5Y cells were plated in 96-well black plates with clear bottoms. After 6 d of administration of RA, serum-free medium containing different concentration of NTU283 (0, 20, 100, and 500 μmol/L) with or without 1 μmol/L Aβ was added to each well. After a 24 h treatment, cells were fixed and permeabilized. Samples were co-incubated with anti-tTG or anti-isopeptide and anti-β-actin antibodies overnight at 4 °C. Then, near-infrared fluorescent second antibodies (LI-COR) were incubated for 1 h at RT. The target proteins and β-actin were double-labeled with different second antibodies (IRDye 680RD, IRDye 800CW) matching their primary antibodies host. Fluorescence signals were detected by Odyssey SA (LI-COR) and analyzed with Image Studio Ver 3.1. All fluorescence densities were quantified and normalized by β-actin. The representative images of tTG (A), isopeptide (B) and relative quantification levels are shown. Statistical data are expressed as the mean±SEM from six independent experiment groups ( n =6). * P

    Journal: Acta Pharmacologica Sinica

    Article Title: Inhibition of tissue transglutaminase promotes Aβ-induced apoptosis in SH-SY5Y cells

    doi: 10.1038/aps.2016.95

    Figure Lengend Snippet: In-cell Western analysis of tTG and isopeptide in NTU283- and Aβ-treated SH-SY5Y cells. SH-SY5Y cells were plated in 96-well black plates with clear bottoms. After 6 d of administration of RA, serum-free medium containing different concentration of NTU283 (0, 20, 100, and 500 μmol/L) with or without 1 μmol/L Aβ was added to each well. After a 24 h treatment, cells were fixed and permeabilized. Samples were co-incubated with anti-tTG or anti-isopeptide and anti-β-actin antibodies overnight at 4 °C. Then, near-infrared fluorescent second antibodies (LI-COR) were incubated for 1 h at RT. The target proteins and β-actin were double-labeled with different second antibodies (IRDye 680RD, IRDye 800CW) matching their primary antibodies host. Fluorescence signals were detected by Odyssey SA (LI-COR) and analyzed with Image Studio Ver 3.1. All fluorescence densities were quantified and normalized by β-actin. The representative images of tTG (A), isopeptide (B) and relative quantification levels are shown. Statistical data are expressed as the mean±SEM from six independent experiment groups ( n =6). * P

    Article Snippet: Anti-Bax, anti-Bcl-2, anti-β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from ProteinTech (Chicago, IL, USA).

    Techniques: In-Cell ELISA, Concentration Assay, Incubation, Labeling, Fluorescence

    Proteomic analysis of the PSD fraction of GluN2B −/− cortical neurons. A , PSDs were successfully purified from high-density rat cortical cultures (14–15 DIV). Western blot for markers of the presynaptic (Synaptophysin) and postsynaptic fractions (PSD-95, SynGAP, NMDAR receptor subunits GluN1 and GluN2A, and AMPAR subunit GluA1) validated PSD isolation of PSDs. Transferrin receptor (Transf R) and β-actin were used as controls. Hom, Homogenate; S1, non-nuclear fraction; P2 crude synaptosomes. Equal amounts (13 μg) of each fraction were applied. B , PSD-localized proteins (PSD-95, GluN1, SynGAP) are increased relative to the homogenate in the PSD fraction, whereas presynaptic synaptophysin (Synapto.) or the trans membrane receptor of transferrin are decreased compared with the P2 fraction, consistent with their absence or low expression, respectively, at the PSD. Actin levels did not change in any of the fractions analyzed. Data are presented as mean ± SEM from two independent experiments. C , Setup of the 8-plex iTRAQ experiments. Isolated PSDs from GluN2B +/+ , GluN2B +/− , and GluN2B −/− cortical neurons, 10 μg each, were digested with trypsin and samples (three wild-types, two heterozygous, and three knock-outs) were tagged with a set of 8-plex iTRAQ reagents. Peptides were pooled together and fractionated by 2D liquid chromatography and subject to tandem mass spectrometric analysis. After using peptides for protein identification, the individual contribution of each sample to each peptide can be measured by the intensity of the reporter ion peaks and used for relative quantification. Example spectra correspond to one of the GluN1 peptides identified in the PSD preparations. D , Chymotrypsin-like proteasome activity was measure in protein extracts from GluN2B +/+ and GluN2B −/− cortical neurons. Proteasome activity is significantly decreased ( p

    Journal: The Journal of Neuroscience

    Article Title: GluN2B-Containing NMDA Receptors Regulate AMPA Receptor Traffic through Anchoring of the Synaptic Proteasome

    doi: 10.1523/JNEUROSCI.3567-14.2015

    Figure Lengend Snippet: Proteomic analysis of the PSD fraction of GluN2B −/− cortical neurons. A , PSDs were successfully purified from high-density rat cortical cultures (14–15 DIV). Western blot for markers of the presynaptic (Synaptophysin) and postsynaptic fractions (PSD-95, SynGAP, NMDAR receptor subunits GluN1 and GluN2A, and AMPAR subunit GluA1) validated PSD isolation of PSDs. Transferrin receptor (Transf R) and β-actin were used as controls. Hom, Homogenate; S1, non-nuclear fraction; P2 crude synaptosomes. Equal amounts (13 μg) of each fraction were applied. B , PSD-localized proteins (PSD-95, GluN1, SynGAP) are increased relative to the homogenate in the PSD fraction, whereas presynaptic synaptophysin (Synapto.) or the trans membrane receptor of transferrin are decreased compared with the P2 fraction, consistent with their absence or low expression, respectively, at the PSD. Actin levels did not change in any of the fractions analyzed. Data are presented as mean ± SEM from two independent experiments. C , Setup of the 8-plex iTRAQ experiments. Isolated PSDs from GluN2B +/+ , GluN2B +/− , and GluN2B −/− cortical neurons, 10 μg each, were digested with trypsin and samples (three wild-types, two heterozygous, and three knock-outs) were tagged with a set of 8-plex iTRAQ reagents. Peptides were pooled together and fractionated by 2D liquid chromatography and subject to tandem mass spectrometric analysis. After using peptides for protein identification, the individual contribution of each sample to each peptide can be measured by the intensity of the reporter ion peaks and used for relative quantification. Example spectra correspond to one of the GluN1 peptides identified in the PSD preparations. D , Chymotrypsin-like proteasome activity was measure in protein extracts from GluN2B +/+ and GluN2B −/− cortical neurons. Proteasome activity is significantly decreased ( p

    Article Snippet: Primary: anti-GluN1 (1:500 WB, #MAB363), anti-GluN2A (1:1000, #AB1555P), anti-GluA1 (1:1000 WB or 1:100 ICC, #AB1504), anti-GluA2 (1:1000 WB or 1:500 ICC, #MAB397), anti-phospho-GluA1 Ser845 (1:1000, #AB5849), anti-phospho-GluA2 Ser880 (1:1000, #07-294), phospho-stargazin (Ser239/240) (1:500, #ab3713), phospho-CaMKII α/β subunit (Thr286/287) (1:1000, #06–881) were from Millipore; anti-GluN1 (1:1500 ICC, #MAb 54.1) was from Invitrogen; N-terminal of GluA1 (1:50) was a kind gift from Dr. Andrew Irving; anti-PSD-95 (D27E11) (1:2000 WB or 1:100 ICC, #3450) and anti-phospho-protein kinase C (PKC) substrates (1:1000, #2261S) were from Cell Signaling Technology; anti-SynGAP (1:1000, #PA1-046) and anti-PSD-95 (1:500, #MA 1.046 C7E3-1B8) were from Affinity Bioreagents; anti-Synaptophysin (1:10000, #101 011) was from Synaptic Systems; anti-CaMKII β (K-19) (1:500, #sc-100366) was from Tebu-Bio, Santa Cruz Biotechnology); CaMKII α (6G9) (1:2000, #C265) was from Sigma-Aldrich; anti-phospho-GluA1 Ser 831 (1:5000, #2041) was from Tocris Bioscience; anti-phospho-GluA1 T840 was a kind gift from R. Huganir (JH2811); anti-Transferrin receptor antibody (1:1000, #13-6800) was from Invitrogen; anti-vesicular glutamate transporter 1 (VGLUT1; 1:5000, #AB5905) was from Millipore; anti-MAP2 (1:5000, #ab5392) was from Abcam; and anti-β-actin (1:5000, #1378996) was from Roche Molecular Biochemicals.

    Techniques: Purification, Western Blot, Isolation, Expressing, Two-dimensional Liquid Chromatography, Activity Assay

    APH1B is involved in modulating stemness in SiHa oncospheres. Notes: ( A ) Heat-map of four stem cell pathway-related genes, WNT5B, APC2, WNT9A, and APH1B. ( B ) Real-time RT-PCR detection of the expression of WNT5B, APC2, WNT9A, and APH1B mRNAs in si-E7 and si-NC SiHa oncospheres. ( C ) Western blot detection of APH1B protein in si-E7 and si-NC SiHa oncospheres. ( D ) Phase-contrast photomicrographs of SiHa oncospheres with APH1B inhibition or overexpression in low-adherence cultures for 7 days. ( E ) Western blot detection of SOX2 and OCT4 proteins in SiHa oncospheres with APH1B inhibition or overexpression. ( F and G ) Growth inhibition of SiHa oncospheres with APH1B inhibition or overexpression. Both were seeded in 96-well plates and treated with paclitaxel or cisplatin at different concentrations (0, 1, 2, 5, 10, 20, 40, 60, 80, 100 nM) for 48 hrs, and cell viability was determined by a modified MTT assay. OD values of each treated group were compared with controls at the same time point. ( H ) Representative photomicrographs of clonal expansion of single oncospheres from SiHa with APH1B inhibition or overexpression in low-adherence cultures over a 7-day period. The cluster of oncospheres after days 1, 3, 5, 7 of culture was measured. Western blot expression levels were normalized to those of β-actin. Error bars and mean with SD were from three independent experiments. * P

    Journal: Cancer Management and Research

    Article Title: High-Risk Human Papillomavirus E7 Maintains Stemness Via APH1B In Cervical Cancer Stem-Cell Like Cells

    doi: 10.2147/CMAR.S194239

    Figure Lengend Snippet: APH1B is involved in modulating stemness in SiHa oncospheres. Notes: ( A ) Heat-map of four stem cell pathway-related genes, WNT5B, APC2, WNT9A, and APH1B. ( B ) Real-time RT-PCR detection of the expression of WNT5B, APC2, WNT9A, and APH1B mRNAs in si-E7 and si-NC SiHa oncospheres. ( C ) Western blot detection of APH1B protein in si-E7 and si-NC SiHa oncospheres. ( D ) Phase-contrast photomicrographs of SiHa oncospheres with APH1B inhibition or overexpression in low-adherence cultures for 7 days. ( E ) Western blot detection of SOX2 and OCT4 proteins in SiHa oncospheres with APH1B inhibition or overexpression. ( F and G ) Growth inhibition of SiHa oncospheres with APH1B inhibition or overexpression. Both were seeded in 96-well plates and treated with paclitaxel or cisplatin at different concentrations (0, 1, 2, 5, 10, 20, 40, 60, 80, 100 nM) for 48 hrs, and cell viability was determined by a modified MTT assay. OD values of each treated group were compared with controls at the same time point. ( H ) Representative photomicrographs of clonal expansion of single oncospheres from SiHa with APH1B inhibition or overexpression in low-adherence cultures over a 7-day period. The cluster of oncospheres after days 1, 3, 5, 7 of culture was measured. Western blot expression levels were normalized to those of β-actin. Error bars and mean with SD were from three independent experiments. * P

    Article Snippet: Antibodies used for Western blotting included anti-SOX2 (#3579, CST), anti-OCT4 (#19857, Abcam), anti-β-actin (#1879-1, Epitomics), anti-mouse HRP (#7076, Cell Signaling Technology), and anti-rabbit HRP (#7074, Cell Signaling Technology).

    Techniques: Quantitative RT-PCR, Expressing, Western Blot, Inhibition, Over Expression, Modification, MTT Assay

    HPV 16 E7 maintains stemness in U2OS oncospheres. Notes: ( A ) Phase-contrast photomicrographs of U2OS cells with HPV16 E7 overexpression in low-adherence culture for 7 days. ( B ) Western blot detection of the expression of SOX2 and OCT4 proteins in U2OS oncospheres with HPV16 E7 overexpression. ( C ) Immunofluorescence images of SOX2 and OCT4 in U2OS oncospheres with HPV16 E7 overexpression using an anti-SOX2/OCT4 (green) antibody. DAPI staining (blue) indicates cell nuclei. Images on the left show cells stained with anti-SOX2/OCT4, images in the middle show cells stained with DAPI, and images on the right are merged anti-SOX2/OCT4 and DAPI. All of the contrast images were taken under the same conditions. ( D ) Growth inhibition of U2OS oncospheres with HPV16 E7 overexpression. Both were seeded in 96-well plates and treated with paclitaxel or cisplatin at different concentrations (0, 1, 2, 5, 10, 20, 40, 60, 80, 100 nM) for 48 hrs, and cell viability was determined by a modified MTT assay. OD values of each treated group were compared with controls at the same time point. ( E ) Representative photomicrographs of clonal expansion of single oncospheres from U2OS with HPV16 E7 overexpression in low-adherence cultures over a 7-day period. The cluster of the oncospheres after days 1, 3, 5, 7 of culture was measured. Western blot expression levels were normalized to those of β-actin. Error bars and mean with SD were from three independent experiments. * P

    Journal: Cancer Management and Research

    Article Title: High-Risk Human Papillomavirus E7 Maintains Stemness Via APH1B In Cervical Cancer Stem-Cell Like Cells

    doi: 10.2147/CMAR.S194239

    Figure Lengend Snippet: HPV 16 E7 maintains stemness in U2OS oncospheres. Notes: ( A ) Phase-contrast photomicrographs of U2OS cells with HPV16 E7 overexpression in low-adherence culture for 7 days. ( B ) Western blot detection of the expression of SOX2 and OCT4 proteins in U2OS oncospheres with HPV16 E7 overexpression. ( C ) Immunofluorescence images of SOX2 and OCT4 in U2OS oncospheres with HPV16 E7 overexpression using an anti-SOX2/OCT4 (green) antibody. DAPI staining (blue) indicates cell nuclei. Images on the left show cells stained with anti-SOX2/OCT4, images in the middle show cells stained with DAPI, and images on the right are merged anti-SOX2/OCT4 and DAPI. All of the contrast images were taken under the same conditions. ( D ) Growth inhibition of U2OS oncospheres with HPV16 E7 overexpression. Both were seeded in 96-well plates and treated with paclitaxel or cisplatin at different concentrations (0, 1, 2, 5, 10, 20, 40, 60, 80, 100 nM) for 48 hrs, and cell viability was determined by a modified MTT assay. OD values of each treated group were compared with controls at the same time point. ( E ) Representative photomicrographs of clonal expansion of single oncospheres from U2OS with HPV16 E7 overexpression in low-adherence cultures over a 7-day period. The cluster of the oncospheres after days 1, 3, 5, 7 of culture was measured. Western blot expression levels were normalized to those of β-actin. Error bars and mean with SD were from three independent experiments. * P

    Article Snippet: Antibodies used for Western blotting included anti-SOX2 (#3579, CST), anti-OCT4 (#19857, Abcam), anti-β-actin (#1879-1, Epitomics), anti-mouse HRP (#7076, Cell Signaling Technology), and anti-rabbit HRP (#7074, Cell Signaling Technology).

    Techniques: Over Expression, Western Blot, Expressing, Immunofluorescence, Staining, Inhibition, Modification, MTT Assay

    HPV 16 E7 maintains stemness in SiHa and Caski oncospheres. Notes: ( A ) Phase-contrast photomicrographs of SiHa and Caski cells with HPV16 E7 knockdown in low-adherence culture for 7 days. ( B ) Western blot detection of the expression of SOX2 and OCT4 proteins in SiHa and Caski oncospheres with HPV16 E7 knockdown. ( C ) Immunofluorescence images of SOX2 and OCT4 in SiHa and Caski oncospheres with HPV16 E7 knockdown using an anti-SOX2/OCT4 (green) antibody. DAPI staining (blue) indicates cell nuclei. Images on the left show cells stained with anti-SOX2/OCT4, images in the middle show cells stained with DAPI, and images on the right are merged anti-SOX2/OCT4 and DAPI. All of the contrast images were taken under the same conditions. ( D ) Growth inhibition of in SiHa and Caski oncospheres with HPV16 E7 knockdown. Both were seeded in 96-well plates and treated with paclitaxel or cisplatin at different concentrations (0, 1, 2, 5, 10, 20, 40, 60, 80, 100 nM) for 48 hrs, and cell viability was determined by a modified MTT assay. OD values of each treated group were compared with controls at the same time point. ( E ) Representative photomicrographs of clonal expansion of single oncospheres from SiHa and Caski with HPV16 E7 knockdown in low-adherence cultures over a 7-day period. The cluster of the oncospheres after days 1, 3, 5, 7 of culture was measured. Western blot expression levels were normalized to those of β-actin. Error bars and mean with SD were from three independent experiments. * P

    Journal: Cancer Management and Research

    Article Title: High-Risk Human Papillomavirus E7 Maintains Stemness Via APH1B In Cervical Cancer Stem-Cell Like Cells

    doi: 10.2147/CMAR.S194239

    Figure Lengend Snippet: HPV 16 E7 maintains stemness in SiHa and Caski oncospheres. Notes: ( A ) Phase-contrast photomicrographs of SiHa and Caski cells with HPV16 E7 knockdown in low-adherence culture for 7 days. ( B ) Western blot detection of the expression of SOX2 and OCT4 proteins in SiHa and Caski oncospheres with HPV16 E7 knockdown. ( C ) Immunofluorescence images of SOX2 and OCT4 in SiHa and Caski oncospheres with HPV16 E7 knockdown using an anti-SOX2/OCT4 (green) antibody. DAPI staining (blue) indicates cell nuclei. Images on the left show cells stained with anti-SOX2/OCT4, images in the middle show cells stained with DAPI, and images on the right are merged anti-SOX2/OCT4 and DAPI. All of the contrast images were taken under the same conditions. ( D ) Growth inhibition of in SiHa and Caski oncospheres with HPV16 E7 knockdown. Both were seeded in 96-well plates and treated with paclitaxel or cisplatin at different concentrations (0, 1, 2, 5, 10, 20, 40, 60, 80, 100 nM) for 48 hrs, and cell viability was determined by a modified MTT assay. OD values of each treated group were compared with controls at the same time point. ( E ) Representative photomicrographs of clonal expansion of single oncospheres from SiHa and Caski with HPV16 E7 knockdown in low-adherence cultures over a 7-day period. The cluster of the oncospheres after days 1, 3, 5, 7 of culture was measured. Western blot expression levels were normalized to those of β-actin. Error bars and mean with SD were from three independent experiments. * P

    Article Snippet: Antibodies used for Western blotting included anti-SOX2 (#3579, CST), anti-OCT4 (#19857, Abcam), anti-β-actin (#1879-1, Epitomics), anti-mouse HRP (#7076, Cell Signaling Technology), and anti-rabbit HRP (#7074, Cell Signaling Technology).

    Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Inhibition, Modification, MTT Assay

    APH1B may participate in E7 maintenance of stemness in SiHa oncospheres. Notes: ( A ) Phase-contrast photomicrographs of SiHa oncospheres with or without HPV16 E7 inhibition and APH1B inhibition in low-adherence cultures for 7 days. ( B ) Western blot detection of SOX2 and OCT4 proteins in SiHa oncospheres with or without HPV16 E7 inhibition and APH1B inhibition in low-adherence cultures for 7 days. ( C ) Growth inhibition of SiHa oncospheres with or without HPV16 E7 inhibition and APH1B inhibition. All sphere cells were seeded in 96-well plates and treated with paclitaxel or cisplatin at different concentrations (0, 1, 2, 5, 10, 20, 40, 60, 80, 100 nM) for 48 hrs, and cell viability was determined by a modified MTT assay. OD values of each treated group were compared with controls at the same time point. ( D ) Proposed model: APH1B may participate in the process of E7 maintaining cellular stemness. Western blot expression levels were normalized to those of β-actin. Measurements of the diameters of colonies are shown in the bar graph. Error bars and mean with SD were from three independent experiments. ** P

    Journal: Cancer Management and Research

    Article Title: High-Risk Human Papillomavirus E7 Maintains Stemness Via APH1B In Cervical Cancer Stem-Cell Like Cells

    doi: 10.2147/CMAR.S194239

    Figure Lengend Snippet: APH1B may participate in E7 maintenance of stemness in SiHa oncospheres. Notes: ( A ) Phase-contrast photomicrographs of SiHa oncospheres with or without HPV16 E7 inhibition and APH1B inhibition in low-adherence cultures for 7 days. ( B ) Western blot detection of SOX2 and OCT4 proteins in SiHa oncospheres with or without HPV16 E7 inhibition and APH1B inhibition in low-adherence cultures for 7 days. ( C ) Growth inhibition of SiHa oncospheres with or without HPV16 E7 inhibition and APH1B inhibition. All sphere cells were seeded in 96-well plates and treated with paclitaxel or cisplatin at different concentrations (0, 1, 2, 5, 10, 20, 40, 60, 80, 100 nM) for 48 hrs, and cell viability was determined by a modified MTT assay. OD values of each treated group were compared with controls at the same time point. ( D ) Proposed model: APH1B may participate in the process of E7 maintaining cellular stemness. Western blot expression levels were normalized to those of β-actin. Measurements of the diameters of colonies are shown in the bar graph. Error bars and mean with SD were from three independent experiments. ** P

    Article Snippet: Antibodies used for Western blotting included anti-SOX2 (#3579, CST), anti-OCT4 (#19857, Abcam), anti-β-actin (#1879-1, Epitomics), anti-mouse HRP (#7076, Cell Signaling Technology), and anti-rabbit HRP (#7074, Cell Signaling Technology).

    Techniques: Inhibition, Western Blot, Modification, MTT Assay, Expressing

    PI3K–Akt–Foxo3a activation in Cbl-b–mediated regulation of Foxp3 + iT reg cells. (A) Phosphorylation of Smad proteins in naive CD4 + T cells from WT, Cbl-b KO, and Cbl-b C373A mice. Naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 in the presence of TGF-β for indicated time periods. Whole-cell lysates were immunoblotted with anti–phospho-(p)-Smad2, or anti–p-Smad3, and reprobed with anti-Smad2/3. Smad2, 60 kD; Smad3, 52 kD. Data are representative of three independent experiments. (B) Naive CD4 + T cells from WT and Cbl-b KO mice were stimulated with anti-CD3 and anti-CD28 together with TGF-β in the absence or presence of the PI3K inhibitor LY294002 (LY), the JNK inhibitor SP600125 (SP), the p38 inhibitor SB203580 (SB), or the calcineurin inhibitor cyclosporine A (Csp A). Foxp3 expression was assessed by FACS analysis after 5 d. The percentages of Foxp3-expressing cells are shown. Data are representative of three independent experiments. (C) Naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 for the indicated time periods. Whole-cell lysates were immunoblotted with anti–p-Akt, anti-Akt, anti–p-Erk, anti-Erk2, and anti–β-actin. Akt, 56 kD; Erk, 42/44 kD; β-actin, 45 kD. Data are representative of at least three independent experiments. (D) Regulation of Foxo3a phosphorylation by Cbl-b E3 ligase. Naive CD4 + T cells from WT and Cbl-b KO mice (top) or WT and Cbl-b C373A mice (bottom) were stimulated with anti-CD3 and anti-CD28 for indicated time periods. Whole-cell lysates were immunoblotted with anti–p-Foxo3a, anti-Foxo3a, and anti–β-actin. Foxo3a, 95 kD; β-actin, 45 kD. Data are representative of at least three independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Transcription factors Foxo3a and Foxo1 couple the E3 ligase Cbl-b to the induction of Foxp3 expression in induced regulatory T cells

    doi: 10.1084/jem.20100004

    Figure Lengend Snippet: PI3K–Akt–Foxo3a activation in Cbl-b–mediated regulation of Foxp3 + iT reg cells. (A) Phosphorylation of Smad proteins in naive CD4 + T cells from WT, Cbl-b KO, and Cbl-b C373A mice. Naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 in the presence of TGF-β for indicated time periods. Whole-cell lysates were immunoblotted with anti–phospho-(p)-Smad2, or anti–p-Smad3, and reprobed with anti-Smad2/3. Smad2, 60 kD; Smad3, 52 kD. Data are representative of three independent experiments. (B) Naive CD4 + T cells from WT and Cbl-b KO mice were stimulated with anti-CD3 and anti-CD28 together with TGF-β in the absence or presence of the PI3K inhibitor LY294002 (LY), the JNK inhibitor SP600125 (SP), the p38 inhibitor SB203580 (SB), or the calcineurin inhibitor cyclosporine A (Csp A). Foxp3 expression was assessed by FACS analysis after 5 d. The percentages of Foxp3-expressing cells are shown. Data are representative of three independent experiments. (C) Naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 for the indicated time periods. Whole-cell lysates were immunoblotted with anti–p-Akt, anti-Akt, anti–p-Erk, anti-Erk2, and anti–β-actin. Akt, 56 kD; Erk, 42/44 kD; β-actin, 45 kD. Data are representative of at least three independent experiments. (D) Regulation of Foxo3a phosphorylation by Cbl-b E3 ligase. Naive CD4 + T cells from WT and Cbl-b KO mice (top) or WT and Cbl-b C373A mice (bottom) were stimulated with anti-CD3 and anti-CD28 for indicated time periods. Whole-cell lysates were immunoblotted with anti–p-Foxo3a, anti-Foxo3a, and anti–β-actin. Foxo3a, 95 kD; β-actin, 45 kD. Data are representative of at least three independent experiments.

    Article Snippet: Anti–β-actin was obtained from MP Biomedicals.

    Techniques: Activation Assay, Mouse Assay, Expressing, FACS

    Foxo1 regulates Foxp3 expression. (A and B) Naive CD4 + T cells from WT and Cbl-b KO mice (A) or WT and Cbl-b C373A mice (B) were stimulated with anti-CD3 and anti-CD28 for indicated time periods. Whole-cell lysates were immunoblotted with anti–p-Foxo1, anti-Foxo1 (75 kD), and anti–β-actin (45 kD). Data are representative of three independent experiments. (C) Naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 for 2 d and transduced with retroviruses encoding Foxo1-specific shRNAs (shRNA1 or shRNA2) or with empty LMP vector (control). After 24 h, TGF-β was added and Foxp3 expression was assessed 3 d later by FACS analysis. Cells were treated with puromycin for last 48 h. The percentages of Foxp3-expressing cells in GFP-positive cells are shown. Data are representative of three independent experiments. (D) Cell lysates in (C) were immunoblotted with anti-Foxo1 (75 kD), anti-Foxo3a (95 kD), and anti-β-actin (45 kD). Data are representative of three independent experiments. (E) Naive CD4 T cells were stimulated with anti-CD3 and anti-CD28 for 2 d and retrovirally transduced with control-IRES-GFP (GFP), Foxo1 WT-IRES-GFP (Foxo1 WT), or Foxo3a WT-IRES-GFP (Foxo3a WT). After infection, TGF-β was added and Foxp3 expression was assessed 3 d later by FACS analysis. The percentages of Foxp3-expressing cells in GFP-positive cells are shown. Data are representative of four independent experiments. (F) Pull-down assay of Foxo1 binding to WT or mutated (Mut3) sequence of the Foxp3 promoter. DNA pull-down assay was performed using biotinylated DNA probes. The precipitates were subjected to SDS-PAGE, followed by immunoblotting with anti-Foxo1 (75 kD). Data are representative of at least three independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Transcription factors Foxo3a and Foxo1 couple the E3 ligase Cbl-b to the induction of Foxp3 expression in induced regulatory T cells

    doi: 10.1084/jem.20100004

    Figure Lengend Snippet: Foxo1 regulates Foxp3 expression. (A and B) Naive CD4 + T cells from WT and Cbl-b KO mice (A) or WT and Cbl-b C373A mice (B) were stimulated with anti-CD3 and anti-CD28 for indicated time periods. Whole-cell lysates were immunoblotted with anti–p-Foxo1, anti-Foxo1 (75 kD), and anti–β-actin (45 kD). Data are representative of three independent experiments. (C) Naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 for 2 d and transduced with retroviruses encoding Foxo1-specific shRNAs (shRNA1 or shRNA2) or with empty LMP vector (control). After 24 h, TGF-β was added and Foxp3 expression was assessed 3 d later by FACS analysis. Cells were treated with puromycin for last 48 h. The percentages of Foxp3-expressing cells in GFP-positive cells are shown. Data are representative of three independent experiments. (D) Cell lysates in (C) were immunoblotted with anti-Foxo1 (75 kD), anti-Foxo3a (95 kD), and anti-β-actin (45 kD). Data are representative of three independent experiments. (E) Naive CD4 T cells were stimulated with anti-CD3 and anti-CD28 for 2 d and retrovirally transduced with control-IRES-GFP (GFP), Foxo1 WT-IRES-GFP (Foxo1 WT), or Foxo3a WT-IRES-GFP (Foxo3a WT). After infection, TGF-β was added and Foxp3 expression was assessed 3 d later by FACS analysis. The percentages of Foxp3-expressing cells in GFP-positive cells are shown. Data are representative of four independent experiments. (F) Pull-down assay of Foxo1 binding to WT or mutated (Mut3) sequence of the Foxp3 promoter. DNA pull-down assay was performed using biotinylated DNA probes. The precipitates were subjected to SDS-PAGE, followed by immunoblotting with anti-Foxo1 (75 kD). Data are representative of at least three independent experiments.

    Article Snippet: Anti–β-actin was obtained from MP Biomedicals.

    Techniques: Expressing, Mouse Assay, Transduction, Plasmid Preparation, FACS, Infection, Pull Down Assay, Binding Assay, Sequencing, SDS Page

    Effects of perivascular adipose tissue (PVAT) on endothelial nitric oxide synthase (eNOS) expression and phosphorylated residues. The aorta homogenates in the presence and absence of PVAT were used to measure eNOS protein expression (n = 26∼27) (A), eNOS phosphorylation on Thr 495  (n = 7∼8) (B) or Ser 1177  (n = 11) (C), and adenosine monophosphate-activated protein kinase (AMPK) expression (n = 5) (D). Blots are representative of the numbers of animals in parentheses. Densitometric mean values were normalized to  β -actin or eNOS protein levels. (−) PVAT, absence of PVAT; (+) PVAT, presence of PVAT.

    Journal: PLoS ONE

    Article Title: Perivascular Adipose Tissue Inhibits Endothelial Function of Rat Aortas via Caveolin-1

    doi: 10.1371/journal.pone.0099947

    Figure Lengend Snippet: Effects of perivascular adipose tissue (PVAT) on endothelial nitric oxide synthase (eNOS) expression and phosphorylated residues. The aorta homogenates in the presence and absence of PVAT were used to measure eNOS protein expression (n = 26∼27) (A), eNOS phosphorylation on Thr 495 (n = 7∼8) (B) or Ser 1177 (n = 11) (C), and adenosine monophosphate-activated protein kinase (AMPK) expression (n = 5) (D). Blots are representative of the numbers of animals in parentheses. Densitometric mean values were normalized to β -actin or eNOS protein levels. (−) PVAT, absence of PVAT; (+) PVAT, presence of PVAT.

    Article Snippet: 10× radio-immunoprecipitation assay (RIPA) lysis buffer was from Millipore Corporate Headquarters (Temecula, CA, USA); bicinchoninic acid (BCA) protein assay kit and Pierce enhanced chemiluminescent (ECL) Western Blotting Substrate were from Thermo Fisher Scientific (Rockford, IL, USA); anti-p-eNOSThr495 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-p-eNOSSer1177 was from Abcam plc (Cambridge, UK); anti-eNOS, anti-adenosine monophosphate-activated protein kinase (anti-AMPK), anti-Cav-1, anti-β -actin, and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG were from BD Transduction Laboratorie (Lexington, KY, USA); HRP conjugated goat anti-rabbit IgG was from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing

    Role of caveolin-1 (Cav-1) in perivascular adipose tissue (PVAT)-enhanced vasocontraction. Western blot analysis of Cav-1 was performed in aorta homogenates in the presence (n = 11) and absence (n = 12) of PVAT (A). Blots are representative of the numbers of animals in parentheses. Densitometric mean values were normalized to  β -actin protein levels. After aortas were preincubated with or without 100 µM methyl- β -cyclodextrin (CD) (n = 10) (B) or 1 mM CD (n = 11) (C), the phenylephrine (PE)-induced vasoconstriction was examined. Nitrate/nitrite levels were measured in aortic homogenates in the presence (n = 13) and absence (n = 12) of PVAT, and the addition of CD (1 mM) reversed the decreased nitrate/nitrite levels caused by PVAT (n = 3) (D). An original trace demonstrates contractile responses to incremental concentrations of PE (1 nM∼10 µM) in the presence and absence of PVAT after pretreatment aorta with CD (100 µM) (E) or 1 mM (F)). Values are means ± SEM for the numbers of animals in parentheses. * P

    Journal: PLoS ONE

    Article Title: Perivascular Adipose Tissue Inhibits Endothelial Function of Rat Aortas via Caveolin-1

    doi: 10.1371/journal.pone.0099947

    Figure Lengend Snippet: Role of caveolin-1 (Cav-1) in perivascular adipose tissue (PVAT)-enhanced vasocontraction. Western blot analysis of Cav-1 was performed in aorta homogenates in the presence (n = 11) and absence (n = 12) of PVAT (A). Blots are representative of the numbers of animals in parentheses. Densitometric mean values were normalized to β -actin protein levels. After aortas were preincubated with or without 100 µM methyl- β -cyclodextrin (CD) (n = 10) (B) or 1 mM CD (n = 11) (C), the phenylephrine (PE)-induced vasoconstriction was examined. Nitrate/nitrite levels were measured in aortic homogenates in the presence (n = 13) and absence (n = 12) of PVAT, and the addition of CD (1 mM) reversed the decreased nitrate/nitrite levels caused by PVAT (n = 3) (D). An original trace demonstrates contractile responses to incremental concentrations of PE (1 nM∼10 µM) in the presence and absence of PVAT after pretreatment aorta with CD (100 µM) (E) or 1 mM (F)). Values are means ± SEM for the numbers of animals in parentheses. * P

    Article Snippet: 10× radio-immunoprecipitation assay (RIPA) lysis buffer was from Millipore Corporate Headquarters (Temecula, CA, USA); bicinchoninic acid (BCA) protein assay kit and Pierce enhanced chemiluminescent (ECL) Western Blotting Substrate were from Thermo Fisher Scientific (Rockford, IL, USA); anti-p-eNOSThr495 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-p-eNOSSer1177 was from Abcam plc (Cambridge, UK); anti-eNOS, anti-adenosine monophosphate-activated protein kinase (anti-AMPK), anti-Cav-1, anti-β -actin, and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG were from BD Transduction Laboratorie (Lexington, KY, USA); HRP conjugated goat anti-rabbit IgG was from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Western Blot

    Hepatic overexpression of HNF4 α  in rats decreased cholesterol levels in both plasma and liver. Rats were intravenously injected with Ad-HNF4 α  or Ad-GFP ( n  = 6), then killed 7 days post-infection. (a) Liver extracts were used to determine protein expression by western blot analysis with antibodies against HNF4 α  or  β -actin. (b) Relative mRNA levels of hepatic HNF4 α  target genes, including G6p, Apoc3, Apob, and L-pk, were examined by qPCR. (c) Plasma cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were determined. (d) Liver cholesterol was  measured in lipid content of rat liver. Data are mean ± SEM, * P

    Journal: PPAR Research

    Article Title: Upregulation of Scavenger Receptor BI by Hepatic Nuclear Factor 4α through a Peroxisome Proliferator-Activated Receptor γ-Dependent Mechanism in Liver

    doi: 10.1155/2011/164925

    Figure Lengend Snippet: Hepatic overexpression of HNF4 α in rats decreased cholesterol levels in both plasma and liver. Rats were intravenously injected with Ad-HNF4 α or Ad-GFP ( n = 6), then killed 7 days post-infection. (a) Liver extracts were used to determine protein expression by western blot analysis with antibodies against HNF4 α or β -actin. (b) Relative mRNA levels of hepatic HNF4 α target genes, including G6p, Apoc3, Apob, and L-pk, were examined by qPCR. (c) Plasma cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were determined. (d) Liver cholesterol was measured in lipid content of rat liver. Data are mean ± SEM, * P

    Article Snippet: Protein expression of sEH, GAPDH and β -actin was detected by use of polyclonal antibodies anti-HNF4α (Santa Cruz Biotechnology, Santa Cruz, Calif, USA), anti-SR-BI (NOVUS Biologicals, Littleton, Colo, USA) and anti-β -actin (Bioss, Beijing, China) followed by horseradish peroxidase-conjugated secondary antibody.

    Techniques: Over Expression, Injection, Infection, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    Expression pattern of hepatic cholesterol-regulatory genes under HNF4 α  overexpression in rat liver. (a) Total RNA was extracted from rat liver and the mRNA levels of hepatic cholesterol-regulatory genes, including HMGCoAR, LCAT, PLTP, LDLR, SR-BI, ABCA1, ABCG5, ABCG8, and CYP7A1, were measured by qPCR. Data are mean ± SEM mRNA levels normalized to that of  β -actin and expressed as fold of GFP-infected group. * P

    Journal: PPAR Research

    Article Title: Upregulation of Scavenger Receptor BI by Hepatic Nuclear Factor 4α through a Peroxisome Proliferator-Activated Receptor γ-Dependent Mechanism in Liver

    doi: 10.1155/2011/164925

    Figure Lengend Snippet: Expression pattern of hepatic cholesterol-regulatory genes under HNF4 α overexpression in rat liver. (a) Total RNA was extracted from rat liver and the mRNA levels of hepatic cholesterol-regulatory genes, including HMGCoAR, LCAT, PLTP, LDLR, SR-BI, ABCA1, ABCG5, ABCG8, and CYP7A1, were measured by qPCR. Data are mean ± SEM mRNA levels normalized to that of β -actin and expressed as fold of GFP-infected group. * P

    Article Snippet: Protein expression of sEH, GAPDH and β -actin was detected by use of polyclonal antibodies anti-HNF4α (Santa Cruz Biotechnology, Santa Cruz, Calif, USA), anti-SR-BI (NOVUS Biologicals, Littleton, Colo, USA) and anti-β -actin (Bioss, Beijing, China) followed by horseradish peroxidase-conjugated secondary antibody.

    Techniques: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Infection

    HNF4 α -upregulated SR-BI depends on PPAR γ . Rat primary hepatocytes were pretreated with or without PPAR γ  antagonist BADGE (10  μ M) for 30 min and then infected with Ad-HNF4 α  or Ad-GFP. (a) Real-time PCR analysis of mRNA expression and (b) western blot analysis of protein expression from 3 independent experiments.  β -actin was used as an internal control. Data are mean ± SEM mRNA levels normalized to that of  β -actin and expressed as fold of control group. * P

    Journal: PPAR Research

    Article Title: Upregulation of Scavenger Receptor BI by Hepatic Nuclear Factor 4α through a Peroxisome Proliferator-Activated Receptor γ-Dependent Mechanism in Liver

    doi: 10.1155/2011/164925

    Figure Lengend Snippet: HNF4 α -upregulated SR-BI depends on PPAR γ . Rat primary hepatocytes were pretreated with or without PPAR γ antagonist BADGE (10  μ M) for 30 min and then infected with Ad-HNF4 α or Ad-GFP. (a) Real-time PCR analysis of mRNA expression and (b) western blot analysis of protein expression from 3 independent experiments. β -actin was used as an internal control. Data are mean ± SEM mRNA levels normalized to that of β -actin and expressed as fold of control group. * P

    Article Snippet: Protein expression of sEH, GAPDH and β -actin was detected by use of polyclonal antibodies anti-HNF4α (Santa Cruz Biotechnology, Santa Cruz, Calif, USA), anti-SR-BI (NOVUS Biologicals, Littleton, Colo, USA) and anti-β -actin (Bioss, Beijing, China) followed by horseradish peroxidase-conjugated secondary antibody.

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

    Hepatic nuclear factor 4 α (HNF4 α ) induced peroxisome proliferator-activated receptor  γ  (PPAR γ ) transcription activity in hepatocytes. (a) Quantitative RT-PCR (qRT-PCR) analysis of mRNA levels of PPAR γ  and its subtypes PPAR γ 1 and PPAR γ 2 in isolated primary hepatocytes. (b) HepG2 cells were cotransfected with plasmids of 3 × PPRE-Luc reporter plasmid with pMT7-HNF4 α  or vehicle plasmid for 24 hr, and luciferase activity with PPAR γ  antagonist BADGE and agonist rosiglitazone (Rosig) was measured. The  β -gal plasmid was cotransfected as a transfection control. Promoter activities were measured by relative luciferase activity, which was normalized to that of  β -gal. Results are mean ± SEM mRNA levels normalized to that of  β -actin and expressed as fold of control group (DMSO). Results are representative of 3 independent experiments. * P

    Journal: PPAR Research

    Article Title: Upregulation of Scavenger Receptor BI by Hepatic Nuclear Factor 4α through a Peroxisome Proliferator-Activated Receptor γ-Dependent Mechanism in Liver

    doi: 10.1155/2011/164925

    Figure Lengend Snippet: Hepatic nuclear factor 4 α (HNF4 α ) induced peroxisome proliferator-activated receptor γ (PPAR γ ) transcription activity in hepatocytes. (a) Quantitative RT-PCR (qRT-PCR) analysis of mRNA levels of PPAR γ and its subtypes PPAR γ 1 and PPAR γ 2 in isolated primary hepatocytes. (b) HepG2 cells were cotransfected with plasmids of 3 × PPRE-Luc reporter plasmid with pMT7-HNF4 α or vehicle plasmid for 24 hr, and luciferase activity with PPAR γ antagonist BADGE and agonist rosiglitazone (Rosig) was measured. The β -gal plasmid was cotransfected as a transfection control. Promoter activities were measured by relative luciferase activity, which was normalized to that of β -gal. Results are mean ± SEM mRNA levels normalized to that of β -actin and expressed as fold of control group (DMSO). Results are representative of 3 independent experiments. * P

    Article Snippet: Protein expression of sEH, GAPDH and β -actin was detected by use of polyclonal antibodies anti-HNF4α (Santa Cruz Biotechnology, Santa Cruz, Calif, USA), anti-SR-BI (NOVUS Biologicals, Littleton, Colo, USA) and anti-β -actin (Bioss, Beijing, China) followed by horseradish peroxidase-conjugated secondary antibody.

    Techniques: Activity Assay, Quantitative RT-PCR, Isolation, Plasmid Preparation, Luciferase, Transfection

    Pulmonary muscarinic receptors function and expression in allergic AIRmin and AIRmax mice. AIRmin or AIRmax mice were sensitized and challenged with OVA as in  Figure 4 . Control group consisted of nonmanipulated animals. The experiments were performed 24 h after the last OVA challenge. Respiratory pattern of allergic AIRmin (a and c) or AIRmax (b and d) to inhaled MCh was evaluated in the presence or absence of gallamine. Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh. Area under the curve was obtained from Penh values (c and d). Gene (e and g) or protein (f and h) expression of the M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from OVA-sensitized AIRmin and AIRmax mice. The real-time PCR was carried out using  β -actin gene expression as internal control for normalization of M3R (e) and M2R (g) mRNA transcription levels. In Western blot analysis the density of M3R (f) and M2R (h) protein expression was normalized to actin expression in each sample. Data are expressed as mean ± SEM of four mice per group and are representative of two experiments. Western blots data were quantified by densitometry using the ImageJ software (NIH). Statistical analyses of Student's  t  test for (a), (b), (e), (f), (g), and (h). Statistical analyses of ANOVA following Tukey HSD for (c) and (d). * P

    Journal: BioMed Research International

    Article Title: Role of M2 Muscarinic Receptor in the Airway Response to Methacholine of Mice Selected for Minimal or Maximal Acute Inflammatory Response

    doi: 10.1155/2013/805627

    Figure Lengend Snippet: Pulmonary muscarinic receptors function and expression in allergic AIRmin and AIRmax mice. AIRmin or AIRmax mice were sensitized and challenged with OVA as in Figure 4 . Control group consisted of nonmanipulated animals. The experiments were performed 24 h after the last OVA challenge. Respiratory pattern of allergic AIRmin (a and c) or AIRmax (b and d) to inhaled MCh was evaluated in the presence or absence of gallamine. Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh. Area under the curve was obtained from Penh values (c and d). Gene (e and g) or protein (f and h) expression of the M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from OVA-sensitized AIRmin and AIRmax mice. The real-time PCR was carried out using β -actin gene expression as internal control for normalization of M3R (e) and M2R (g) mRNA transcription levels. In Western blot analysis the density of M3R (f) and M2R (h) protein expression was normalized to actin expression in each sample. Data are expressed as mean ± SEM of four mice per group and are representative of two experiments. Western blots data were quantified by densitometry using the ImageJ software (NIH). Statistical analyses of Student's t test for (a), (b), (e), (f), (g), and (h). Statistical analyses of ANOVA following Tukey HSD for (c) and (d). * P

    Article Snippet: The following polyclonal rabbit antisera were used: anti-M1 (1 : 200), anti-M2 (1 : 100), anti-M3 (1 : 100), and anti-β -actin (1 : 400) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). β -actin protein expression was used as an internal standard for relative quantification of muscarinic receptors expression levels.

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

    Respiratory pattern and expression of muscarinic receptors in AIRmin and AIRmax mice. Respiratory pattern was determined in awake, unrestrained mice by noninvasive whole-body barometric plethysmography. (a) Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh (3, 6, 12, and 25 mg/mL) and (b) provocative concentration of aerosol MCh at a 200% increase (PC200) over baseline values. Gene (c and e) or protein (d and f) expression of M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from AIRmin and AIRmax mice. Real-time PCR was carried out using  β -actin gene expression as internal control for normalization of M3R (c) and M2R (e) mRNA transcription levels. All PCR reactions were quantitative reactions made by real-time PCR. In Western blot analysis the density of M3R (d) and M2R (f) protein expression was nor aerosol at a malized to actin expression in each sample. Western blots were quantified by densitometry using the ImageJ software (NIH). Real-time PCR and Western-blot analyses were performed using pooled lungs from 5 mice. Data are expressed as mean ± SEM of five mice per group and are representative of three experiments; * P

    Journal: BioMed Research International

    Article Title: Role of M2 Muscarinic Receptor in the Airway Response to Methacholine of Mice Selected for Minimal or Maximal Acute Inflammatory Response

    doi: 10.1155/2013/805627

    Figure Lengend Snippet: Respiratory pattern and expression of muscarinic receptors in AIRmin and AIRmax mice. Respiratory pattern was determined in awake, unrestrained mice by noninvasive whole-body barometric plethysmography. (a) Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh (3, 6, 12, and 25 mg/mL) and (b) provocative concentration of aerosol MCh at a 200% increase (PC200) over baseline values. Gene (c and e) or protein (d and f) expression of M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from AIRmin and AIRmax mice. Real-time PCR was carried out using β -actin gene expression as internal control for normalization of M3R (c) and M2R (e) mRNA transcription levels. All PCR reactions were quantitative reactions made by real-time PCR. In Western blot analysis the density of M3R (d) and M2R (f) protein expression was nor aerosol at a malized to actin expression in each sample. Western blots were quantified by densitometry using the ImageJ software (NIH). Real-time PCR and Western-blot analyses were performed using pooled lungs from 5 mice. Data are expressed as mean ± SEM of five mice per group and are representative of three experiments; * P

    Article Snippet: The following polyclonal rabbit antisera were used: anti-M1 (1 : 200), anti-M2 (1 : 100), anti-M3 (1 : 100), and anti-β -actin (1 : 400) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). β -actin protein expression was used as an internal standard for relative quantification of muscarinic receptors expression levels.

    Techniques: Expressing, Mouse Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot, Polymerase Chain Reaction, Software

    DBT regulates expressions of Bcl2 and Bax. Cultured cortical neurons were co-treated with various concentration of DBT (DBT-L: 12.5 μg/mL; DBT-H: 100 μg/mL) and Aβ (20 μM) for 48 h. The protein expression levels of Bcl2 (~ 27 kDa) and Bax (~ 21 kDa) were detected by immunoblot analysis using specific antibodies, and β-actin (~ 42 kDa) served as an internal control. Quantification of target protein expression was calculated by a densitometer. Values were expressed as the percentage of change as compared to control reading, in Mean ± SEM, where n = 3. Statistically significant changes were classified as more significant (**) where p

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Danggui Buxue Tang, an ancient Chinese herbal decoction, protects β-amyloid-induced cell death in cultured cortical neurons

    doi: 10.1186/s12906-018-2411-6

    Figure Lengend Snippet: DBT regulates expressions of Bcl2 and Bax. Cultured cortical neurons were co-treated with various concentration of DBT (DBT-L: 12.5 μg/mL; DBT-H: 100 μg/mL) and Aβ (20 μM) for 48 h. The protein expression levels of Bcl2 (~ 27 kDa) and Bax (~ 21 kDa) were detected by immunoblot analysis using specific antibodies, and β-actin (~ 42 kDa) served as an internal control. Quantification of target protein expression was calculated by a densitometer. Values were expressed as the percentage of change as compared to control reading, in Mean ± SEM, where n = 3. Statistically significant changes were classified as more significant (**) where p

    Article Snippet: The membranes were incubated with anti-Bcl2, Bax, cleaved- PARP and cleaved-caspase 3/9 (CST, Danvers, MA) at 1: 1000 dilutions, respectively, and anti-β-actin (Abcam, Cambridge, MA) at 1: 10,000 dilutions were kept for over 12 h at cold room.

    Techniques: Cell Culture, Concentration Assay, Expressing

    SMARCAD1 enhances proliferation of PANC-1 cells. A-B. The efficiency of SMARCAD1 knockdown (A) or overexpression (B) in PANC-1 cells was detected by western blotting. β-actin was used as an internal control. C-D. CCK8 assay was performed to determine the proliferation of PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) at the indicated time points after plated. Cell viability was measured at 450nm. E-F. The effect of SMARCAD1 knockdown (E) or overexpression (F) on Colony-forming of PANC-1 cells was shown in the top panels. Number of foci was counted as shown in the bottom panels. All data were presented as mean ±SEM. *p

    Journal: International Journal of Biological Sciences

    Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

    doi: 10.7150/ijbs.29562

    Figure Lengend Snippet: SMARCAD1 enhances proliferation of PANC-1 cells. A-B. The efficiency of SMARCAD1 knockdown (A) or overexpression (B) in PANC-1 cells was detected by western blotting. β-actin was used as an internal control. C-D. CCK8 assay was performed to determine the proliferation of PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) at the indicated time points after plated. Cell viability was measured at 450nm. E-F. The effect of SMARCAD1 knockdown (E) or overexpression (F) on Colony-forming of PANC-1 cells was shown in the top panels. Number of foci was counted as shown in the bottom panels. All data were presented as mean ±SEM. *p

    Article Snippet: Anti-bodies used are as follows: Anti-SMARCAD1 (CST, 12458), anti-β-actin (Transgen Biotech, Hc201), anti-E-cadherin (CST, 14472), anti-N-cadherin (BD, 610920), anti-slug (CST, 9585), anti-β-catenin (CST, 9562),anti-MMP9 (CST, 2270), anti-MMP2 (ABclonal, A6247), anti-c-Myc (CST, 9402), anti-Cyclin D1 (CST, 2978), anti-survivin (Sigma, ab8228).

    Techniques: Over Expression, Western Blot, CCK-8 Assay

    Induction of apoptotic and autophagic cell death in TMZ-resistant glioma cells by VPA and TMZ combination. T98 and U138 cells were untreated or treated with VPA, TMZ, or their combination. (a) The expression of MGMT, Bcl-2, Bak, and caspase-3 was evaluated using western blotting. (b) Conversion of LC3-I to LC3-II was also determined by western blotting.  β -actin was used as a loading control. The results are representative of three independent experiments.

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Valproic Acid Downregulates the Expression of MGMT and Sensitizes Temozolomide-Resistant Glioma Cells

    doi: 10.1155/2012/987495

    Figure Lengend Snippet: Induction of apoptotic and autophagic cell death in TMZ-resistant glioma cells by VPA and TMZ combination. T98 and U138 cells were untreated or treated with VPA, TMZ, or their combination. (a) The expression of MGMT, Bcl-2, Bak, and caspase-3 was evaluated using western blotting. (b) Conversion of LC3-I to LC3-II was also determined by western blotting. β -actin was used as a loading control. The results are representative of three independent experiments.

    Article Snippet: Western Blot Analysis Antibodies were obtained from commercial sources: anti-MGMT from Cell Signaling Technology (Danvers, MA, USA), anti-β -actin from Applied Biological Materials (Richmond, BC, Canada), anti-LC3B from Sigma, and anti-Bcl-2, anti-Bak, and anti-caspase-3 from New England Biolabs (Ipswich, MA, USA).

    Techniques: Expressing, Western Blot

    Effect of VPA on the expression of MGMT in TMZ-resistant glioma cells. (a) Cell lysates of four human glioma cell lines were subjected to western blotting using an anti-MGMT antibody. This revealed an absence of MGMT expression in the U87 and U251 cell lines. In contrast, the other two cell lines, T98 and U138, exhibited MGMT expression at the protein level. (b) MGMT expression in the T98 and U138 TMZ-resistant cells treated with varying concentrations of VPA (0–8 mM).  β -Actin was used as a loading control. (c) Cells were exposed to medium containing various concentrations of VPA (0–4 mM) with 50  μ M TMZ for 72 h and were measured using MTT assay. Columns, mean; bars, SE. * P

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Valproic Acid Downregulates the Expression of MGMT and Sensitizes Temozolomide-Resistant Glioma Cells

    doi: 10.1155/2012/987495

    Figure Lengend Snippet: Effect of VPA on the expression of MGMT in TMZ-resistant glioma cells. (a) Cell lysates of four human glioma cell lines were subjected to western blotting using an anti-MGMT antibody. This revealed an absence of MGMT expression in the U87 and U251 cell lines. In contrast, the other two cell lines, T98 and U138, exhibited MGMT expression at the protein level. (b) MGMT expression in the T98 and U138 TMZ-resistant cells treated with varying concentrations of VPA (0–8 mM). β -Actin was used as a loading control. (c) Cells were exposed to medium containing various concentrations of VPA (0–4 mM) with 50  μ M TMZ for 72 h and were measured using MTT assay. Columns, mean; bars, SE. * P

    Article Snippet: Western Blot Analysis Antibodies were obtained from commercial sources: anti-MGMT from Cell Signaling Technology (Danvers, MA, USA), anti-β -actin from Applied Biological Materials (Richmond, BC, Canada), anti-LC3B from Sigma, and anti-Bcl-2, anti-Bak, and anti-caspase-3 from New England Biolabs (Ipswich, MA, USA).

    Techniques: Expressing, Western Blot, MTT Assay

    Low dose of apelin-36 attenuates cerebral I/R injury-induced caspase-3 activation in rats. (A) The MCA territory of sham treated rats, I/R model rats and I/R model rats treated with apelin-36 (labeled I/R-Apelin) were lysed in RIPA-DOC buffer. Cell lysates were resolved on 12% Tris–Glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cleaved caspase-3 was detected by cleaved caspase-3 antibody and β-actin served as an internal control was detected by β-actin antibody. (B) Quantification of cleaved caspase-3 expression. The ratio of cleaved caspase-3 to β-actin was further normalized to the sham treated rats. Values represent mean ± SEM. N = 5, * p

    Journal: Frontiers in Neurology

    Article Title: Low Dose of Apelin-36 Attenuates ER Stress-Associated Apoptosis in Rats with Ischemic Stroke

    doi: 10.3389/fneur.2017.00556

    Figure Lengend Snippet: Low dose of apelin-36 attenuates cerebral I/R injury-induced caspase-3 activation in rats. (A) The MCA territory of sham treated rats, I/R model rats and I/R model rats treated with apelin-36 (labeled I/R-Apelin) were lysed in RIPA-DOC buffer. Cell lysates were resolved on 12% Tris–Glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cleaved caspase-3 was detected by cleaved caspase-3 antibody and β-actin served as an internal control was detected by β-actin antibody. (B) Quantification of cleaved caspase-3 expression. The ratio of cleaved caspase-3 to β-actin was further normalized to the sham treated rats. Values represent mean ± SEM. N = 5, * p

    Article Snippet: The membranes were blocked with 5% skim milk in TBST for 4 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: anti-cleaved caspase-3 (3:5,000, Wanlei), anti-GRP78 (1:1,000; Cell Signaling), anti-CHOP (3:5,000; Wanlei), and anti-β-actin (1:2,000; Zhongshan Golden Bridge Biotechnology).

    Techniques: Activation Assay, Labeling, Polyacrylamide Gel Electrophoresis, Expressing

    Low dose of apelin-36 inhibits cerebral I/R injury-induced GRP78 and CHOP elevation in rats. (A) Rats were subjected to vehicle or apelin-36 treatment at 2 h after middle cerebral artery occlusion (MCAO) procedure. The brain lysates of MCA territory were resolved on 8–12% Tris–Glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. GRP78 and CHOP were detected by GRP78 and CHOP antibodies, respectively. β-actin served as an internal control was detected by β-actin antibody. (B) and (C) Quantification of CHOP and GRP78 protein expression, respectively. Apelin represents apelin-36. (D) Rats were subjected to vehicle or apelin-36 treatment at 2 h after MCAO procedure. The mRNA of MCA territory was extracted and RT-PCR was performed. The PCR products of GRP78 and CHOP were resolved on 1.5% agarose gel, respectively. β-actin was served as an internal control. (E,F) Quantification of CHOP and GRP78 mRNA expression, respectively. Apelin represents apelin-36. Values represent mean ± SEM. N = 5, * p

    Journal: Frontiers in Neurology

    Article Title: Low Dose of Apelin-36 Attenuates ER Stress-Associated Apoptosis in Rats with Ischemic Stroke

    doi: 10.3389/fneur.2017.00556

    Figure Lengend Snippet: Low dose of apelin-36 inhibits cerebral I/R injury-induced GRP78 and CHOP elevation in rats. (A) Rats were subjected to vehicle or apelin-36 treatment at 2 h after middle cerebral artery occlusion (MCAO) procedure. The brain lysates of MCA territory were resolved on 8–12% Tris–Glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. GRP78 and CHOP were detected by GRP78 and CHOP antibodies, respectively. β-actin served as an internal control was detected by β-actin antibody. (B) and (C) Quantification of CHOP and GRP78 protein expression, respectively. Apelin represents apelin-36. (D) Rats were subjected to vehicle or apelin-36 treatment at 2 h after MCAO procedure. The mRNA of MCA territory was extracted and RT-PCR was performed. The PCR products of GRP78 and CHOP were resolved on 1.5% agarose gel, respectively. β-actin was served as an internal control. (E,F) Quantification of CHOP and GRP78 mRNA expression, respectively. Apelin represents apelin-36. Values represent mean ± SEM. N = 5, * p

    Article Snippet: The membranes were blocked with 5% skim milk in TBST for 4 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: anti-cleaved caspase-3 (3:5,000, Wanlei), anti-GRP78 (1:1,000; Cell Signaling), anti-CHOP (3:5,000; Wanlei), and anti-β-actin (1:2,000; Zhongshan Golden Bridge Biotechnology).

    Techniques: Polyacrylamide Gel Electrophoresis, Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Sch A induces Nrf2 activation in tissues. Protein expression of Nrf2, HO-1, and NQO-1 (A) was evaluated by Western blotting analyses. (B-D) Quantification of Nrf2, HO-1, and NQO-1 levels normalized to that of β-actin. All data are mean ± SD. (##P

    Journal: American Journal of Translational Research

    Article Title: Schizandrin A protects against cerebral ischemia-reperfusion injury by suppressing inflammation and oxidative stress and regulating the AMPK/Nrf2 pathway regulation

    doi:

    Figure Lengend Snippet: Sch A induces Nrf2 activation in tissues. Protein expression of Nrf2, HO-1, and NQO-1 (A) was evaluated by Western blotting analyses. (B-D) Quantification of Nrf2, HO-1, and NQO-1 levels normalized to that of β-actin. All data are mean ± SD. (##P

    Article Snippet: Anti-β-actin and anti-histone H3 were obtained from Beyotime Biotechnology (Shanghai, China).

    Techniques: Activation Assay, Expressing, Western Blot

    Photoreceptor protein expression in GCAP1 −/− retina. A. Immunoblots of SDS polyacrylamide gels containing samples from WT and GCAP1 KO retinas probed with antibodies raised against GCAP2, RetGC1, RetGC2, rod α-transducin (Gtα1), PDE6, arrestin 1 (ARR), GRK1, RGS9, and β-actin, as indicated. B. Average (± SD) integrated chemiluminescence signal intensity in the band for the corresponding antigen in GCAP1 −/− retina relative to the WT for GCAP1 (n = 5), GCAP2 (n = 7), RetGC1 (n = 3), RetGC2 (n = 3), rod α-transducin (n = 3), PDE6 (n = 3), arrestin (n = 3), GRK1 (n = 3), RGS9 (n = 3), and β-actin (n = 3). When compared by one-way ANOVA with Bonferroni’s post hoc test (alpha = 0.01), there were significant differences found in GCAP1 (**) and GCAP2 (*) contents (P

    Journal: PLoS ONE

    Article Title: Enzymatic Relay Mechanism Stimulates Cyclic GMP Synthesis in Rod Photoresponse: Biochemical and Physiological Study in Guanylyl Cyclase Activating Protein 1 Knockout Mice

    doi: 10.1371/journal.pone.0047637

    Figure Lengend Snippet: Photoreceptor protein expression in GCAP1 −/− retina. A. Immunoblots of SDS polyacrylamide gels containing samples from WT and GCAP1 KO retinas probed with antibodies raised against GCAP2, RetGC1, RetGC2, rod α-transducin (Gtα1), PDE6, arrestin 1 (ARR), GRK1, RGS9, and β-actin, as indicated. B. Average (± SD) integrated chemiluminescence signal intensity in the band for the corresponding antigen in GCAP1 −/− retina relative to the WT for GCAP1 (n = 5), GCAP2 (n = 7), RetGC1 (n = 3), RetGC2 (n = 3), rod α-transducin (n = 3), PDE6 (n = 3), arrestin (n = 3), GRK1 (n = 3), RGS9 (n = 3), and β-actin (n = 3). When compared by one-way ANOVA with Bonferroni’s post hoc test (alpha = 0.01), there were significant differences found in GCAP1 (**) and GCAP2 (*) contents (P

    Article Snippet: Antibody against RGS9 was received from Dr. Vladlen Slepak (University of Miami), anti-arrestin 1 antibody was received from Dr. Vsevolod Gurevich (Vanderbilt University), and anti-GRK1 antibody 41072 was received from Dr. Jason Chen (Virginia Commonwealth University); anti-Gtα1 and anti-PDE6α antibodies were from AbCam, anti-β-actin – from GeneTex, and anti-rhodopsin – was from Chemicon/Millipore.

    Techniques: Expressing, Western Blot

    Effects of NCBAE on 2K1C-induced apoptosis. (a) The expression of Bcl-2 and cleaved caspase-3 protein was determined by western blotting. (b) Protein levels were measured by densitometry and presented as histograms. Bcl-2 and cleaved caspase-3 protein normalized to  β -actin.  ∗ P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: The Effects of Aqueous Extract from Nardostachys chinensis Batalin on Blood Pressure and Cardiac Hypertrophy in Two-Kidney One-Clip Hypertensive Rats

    doi: 10.1155/2017/4031950

    Figure Lengend Snippet: Effects of NCBAE on 2K1C-induced apoptosis. (a) The expression of Bcl-2 and cleaved caspase-3 protein was determined by western blotting. (b) Protein levels were measured by densitometry and presented as histograms. Bcl-2 and cleaved caspase-3 protein normalized to β -actin. ∗ P

    Article Snippet: The membranes were incubated overnight at 4°C with the rabbit anti-rat monoclonal antibodies of anti-Bcl-2 (1 : 5000; Abcam® Inc., Cambridge, England) and polyclonal antibodies of anti-caspase-3 (1 : 1000; Cell Signaling Technology Inc., Danvers, USA) and anti-β -actin (1 : 5000; Bioworld Technology Inc., Minnesota, USA), respectively.

    Techniques: Expressing, Western Blot

    (a) Western blotting for EGFR (170 kDa) and Beclin1 (60 kDa) in representative H-EGFR/L-Beclin1 (lanes 1 and 2), L-EGFR/H-Beclin1 (lanes 3–5), H-EGFR/H-Beclin1 (lanes 6–8), and L-EGFR/L-Beclin1 (lanes 9–11) GBs. (b) Up- or downregulation of either EGFR or Beclin1 is quantified by densitometric data analysis, which shows the relative expression of EGFR and Beclin1 after normalization to the  β -actin bands. Data are reported as means ± S.E. of three densitometric analyses of the same sample.

    Journal: BioMed Research International

    Article Title: Combined Epidermal Growth Factor Receptor and Beclin1 Autophagic Protein Expression Analysis Identifies Different Clinical Presentations, Responses to Chemo- and Radiotherapy, and Prognosis in Glioblastoma

    doi: 10.1155/2015/208076

    Figure Lengend Snippet: (a) Western blotting for EGFR (170 kDa) and Beclin1 (60 kDa) in representative H-EGFR/L-Beclin1 (lanes 1 and 2), L-EGFR/H-Beclin1 (lanes 3–5), H-EGFR/H-Beclin1 (lanes 6–8), and L-EGFR/L-Beclin1 (lanes 9–11) GBs. (b) Up- or downregulation of either EGFR or Beclin1 is quantified by densitometric data analysis, which shows the relative expression of EGFR and Beclin1 after normalization to the β -actin bands. Data are reported as means ± S.E. of three densitometric analyses of the same sample.

    Article Snippet: Membranes were blocked with 3% nonfat milk (BioRad) in PBS Tween 0.05% (PBST) and incubated overnight at 4°C with the following antibodies: anti-EGFR (undiluted, Novocastra), anti-Beclin1 (1 : 270, Sigma-Aldrich), and anti-β -actin (1 : 500, catalogue number: 04-1116, MERCK Millipore Corporation, Billerica, MA, US) diluted with 3% nonfat milk in PBST.

    Techniques: Western Blot, Expressing

    Nitrogen or carbon starvation induces autophagy in C. neoformans . (A,B) Analysis of GFP-Atg8 processing by western blotting. H99 and atg Δ strains transformed with a plasmid expressing the EGFP-Atg8 fusion protein were collected after shifting to nitrogen starvation medium (SD-N) (A) or carbon starvation medium (SD-C) (B) at the indicated times. Total proteins were extracted and subjected to immunoblot analysis; β-actin was used as the loading control. (C) Localization of EGFP-Atg8 upon nutrient starvation. H99, atg1 Δ and atg13 Δ cells in log phase (starved; 0 h) or after shifting to SD-N or SD-C medium containing 10 μg/mL nocodazole for 4 h were observed by both fluorescence microscopy and differential interferential contrast (DIC) with a laser scanning confocal microscope. (D) Autophagic body formation. H99 and atg Δ strains were incubated in SD-N or SD-C medium containing 1 mM PMSF and 10 μg/mL nocodazole for 4 h and subjected to DIC microscopy. Arrows indicate autophagic bodies within vacuoles.

    Journal: Frontiers in Microbiology

    Article Title: Conserved Autophagy Pathway Contributes to Stress Tolerance and Virulence and Differentially Controls Autophagic Flux Upon Nutrient Starvation in Cryptococcus neoformans

    doi: 10.3389/fmicb.2019.02690

    Figure Lengend Snippet: Nitrogen or carbon starvation induces autophagy in C. neoformans . (A,B) Analysis of GFP-Atg8 processing by western blotting. H99 and atg Δ strains transformed with a plasmid expressing the EGFP-Atg8 fusion protein were collected after shifting to nitrogen starvation medium (SD-N) (A) or carbon starvation medium (SD-C) (B) at the indicated times. Total proteins were extracted and subjected to immunoblot analysis; β-actin was used as the loading control. (C) Localization of EGFP-Atg8 upon nutrient starvation. H99, atg1 Δ and atg13 Δ cells in log phase (starved; 0 h) or after shifting to SD-N or SD-C medium containing 10 μg/mL nocodazole for 4 h were observed by both fluorescence microscopy and differential interferential contrast (DIC) with a laser scanning confocal microscope. (D) Autophagic body formation. H99 and atg Δ strains were incubated in SD-N or SD-C medium containing 1 mM PMSF and 10 μg/mL nocodazole for 4 h and subjected to DIC microscopy. Arrows indicate autophagic bodies within vacuoles.

    Article Snippet: The membrane was blocked by incubation in 5% (w/v) skim milk at 25°C for 2 h. To detect the GFP-Atg8 and internal control, membrane was incubated with anti-GFP (1:3,000; Santa Cruz Biotechnology) or anti-β-actin (1:3,000; GenScript) primary antibody, followed by anti-mouse IgG HRP-linked secondary antibody (1:5,000; Cell Signal).

    Techniques: Western Blot, Transformation Assay, Plasmid Preparation, Expressing, Fluorescence, Microscopy, Incubation

    IL-5 and CD40L stimulation expands a population of B cells enriched for FasL expression. ( A ) Diagram of B cell co-culture experiments with CD40L-expressing fibroblasts for study of the effects of IL-5 on B cell growth and function. ( B ) CD19 + B cells were cultured as illustrated in (A) and harvested after five days. Viable B cells recovered from culture were quantified from replicate samples using a hemocytometer and trypan blue exclusion. The dotted line represents the concentration of B cells at the beginning of the experiment. ( C ) Proliferation of B cells cultured with CD40L-expressing fibroblasts in the presence or absence of IL-5 was assessed by the incorporation of 3 H-thymidine. ( D ) B cells stimulated with CD40L in the presence or absence of IL-5 were stained for FasL, CD5, and CD1d and compared with freshly-isolated CD19 + B cells. ( E ) Median fluorescence intensity of surface markers stained as in (D) on freshly-isolated CD19 + B cells, B cells stimulated with CD40L, and B cells stimulated with CD40L and IL-5. ( F ) The frequency of FasL + B cells among freshly-isolated CD19 + B cells and B cells stimulated with CD40L and IL-5 was measured by flow cytometry as in (D). ( G ) Cell lysates from equal numbers of freshly-isolated CD19 + B cells and IL-5-stimulated B cells were probed by immunoblot for FasL and β-Actin. Data are representative of more than four independent experiments. *** p

    Journal: PLoS ONE

    Article Title: Interleukin-5 Supports the Expansion of Fas Ligand-Expressing Killer B Cells that Induce Antigen-Specific Apoptosis of CD4+ T Cells and Secrete Interleukin-10

    doi: 10.1371/journal.pone.0070131

    Figure Lengend Snippet: IL-5 and CD40L stimulation expands a population of B cells enriched for FasL expression. ( A ) Diagram of B cell co-culture experiments with CD40L-expressing fibroblasts for study of the effects of IL-5 on B cell growth and function. ( B ) CD19 + B cells were cultured as illustrated in (A) and harvested after five days. Viable B cells recovered from culture were quantified from replicate samples using a hemocytometer and trypan blue exclusion. The dotted line represents the concentration of B cells at the beginning of the experiment. ( C ) Proliferation of B cells cultured with CD40L-expressing fibroblasts in the presence or absence of IL-5 was assessed by the incorporation of 3 H-thymidine. ( D ) B cells stimulated with CD40L in the presence or absence of IL-5 were stained for FasL, CD5, and CD1d and compared with freshly-isolated CD19 + B cells. ( E ) Median fluorescence intensity of surface markers stained as in (D) on freshly-isolated CD19 + B cells, B cells stimulated with CD40L, and B cells stimulated with CD40L and IL-5. ( F ) The frequency of FasL + B cells among freshly-isolated CD19 + B cells and B cells stimulated with CD40L and IL-5 was measured by flow cytometry as in (D). ( G ) Cell lysates from equal numbers of freshly-isolated CD19 + B cells and IL-5-stimulated B cells were probed by immunoblot for FasL and β-Actin. Data are representative of more than four independent experiments. *** p

    Article Snippet: Membranes were blocked and incubated with polyclonal rabbit anti-FasL IgG (Millipore) or anti-β-Actin (Cell Signaling).

    Techniques: Expressing, Co-Culture Assay, Cell Culture, Concentration Assay, Staining, Isolation, Fluorescence, Flow Cytometry, Cytometry

    IL-5-mediated induction of killer B cell function, but not FasL expression, is inhibited by IL-4. CD19 + B cells from naïve mice were cultured for five days with CD40L Fb and IL-4, IL-5, or both cytokines concurrently. ( A ) The ability of B cells stimulated under these conditions to induce apoptosis in activated CD4 + T cells was then assessed as in Figure 6 . Apoptotic cells were identified and enumerated (mean ± SEM) on the basis of positive staining for Annexin V by flow cytometry. ( B ) B cells were cultured with CD40L-expressing fibroblasts in the presence or absence of IL-4 and IL-5, or in the presence of both cytokines. Cells were harvested after 5 days in culture and cell lysates were probed for FasL and β-Actin proteins as in Figure 5G . *** p

    Journal: PLoS ONE

    Article Title: Interleukin-5 Supports the Expansion of Fas Ligand-Expressing Killer B Cells that Induce Antigen-Specific Apoptosis of CD4+ T Cells and Secrete Interleukin-10

    doi: 10.1371/journal.pone.0070131

    Figure Lengend Snippet: IL-5-mediated induction of killer B cell function, but not FasL expression, is inhibited by IL-4. CD19 + B cells from naïve mice were cultured for five days with CD40L Fb and IL-4, IL-5, or both cytokines concurrently. ( A ) The ability of B cells stimulated under these conditions to induce apoptosis in activated CD4 + T cells was then assessed as in Figure 6 . Apoptotic cells were identified and enumerated (mean ± SEM) on the basis of positive staining for Annexin V by flow cytometry. ( B ) B cells were cultured with CD40L-expressing fibroblasts in the presence or absence of IL-4 and IL-5, or in the presence of both cytokines. Cells were harvested after 5 days in culture and cell lysates were probed for FasL and β-Actin proteins as in Figure 5G . *** p

    Article Snippet: Membranes were blocked and incubated with polyclonal rabbit anti-FasL IgG (Millipore) or anti-β-Actin (Cell Signaling).

    Techniques: Cell Function Assay, Expressing, Mouse Assay, Cell Culture, Staining, Flow Cytometry, Cytometry