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

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

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

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

Journal: Redox Biology

Article Title: Probucol ameliorates renal injury in diabetic nephropathy by inhibiting the expression of the redox enzyme p66Shc

doi: 10.1016/j.redox.2017.07.002

Figure Lengend Snippet: p-AMPK, SIRT1, AcH3 and p66Shc expression in HK-2 cells exposed to High glucose (HG) conditions and probucol treatment. Panel A–C: Representative Western blots and densitometric analyses of p-AMPK, Sirt1, Ac-H3 and p66Shc expression in HK-2 cells. High glucose (HG) reduced p-AMPK expression in HK-2 cells in a time-dependent manner (A). This effect was reversed by probucol treatment (B). HG notably decreased Sirt1 expression and increased the ration of Ac-H3/H3 and p66Shc expression. These changes were reversed following probucol treatment (C). Densitometric analyses represent four independent experiments and depict the ratios of p-AMPK/total AMPK (A and B), SIRT1/β-actin (D), p66Shc/β-actin (F) and AcH3/H3 (E). G: Immunofluorescence shows decreased Sirt1 expression in HK-2 cells exposed to HG compared to that in LG. This effect was partially blocked by probucol treatment (upper panel). Contrasting results were observed regarding p66Shc expression (middle panel). No changes were observed in DMSO-treated cells. H: ELISA assess showed that the p-AMPK concentrations in HK-2 cells in various groups. I: Sirt1 activity in HK-2 cells. Values are the mean ± SEM (n = 5). *P

Article Snippet: Other materials, including an In situ BrdU-Red DNA Fragmentation (TUNEL) Assay Kit (ab66110) and anti-β-actin antibody (ab8226), were purchased from Abcam (UK).

Techniques: Expressing, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Redox Biology

Article Title: Probucol ameliorates renal injury in diabetic nephropathy by inhibiting the expression of the redox enzyme p66Shc

doi: 10.1016/j.redox.2017.07.002

Figure Lengend Snippet: Renal Sirt1, Ac-H3, p66Shc and p-AMPK expression in STZ-induced diabetic mice following probucol treatment. Panel A–B: Western blot analysis of p-AMPK and total AMPK (A), Sirt1 (B, upper panel), Ac-H3 Total H3 (B, middle panel) and p66Shc (B, bottom panel) protein expression in the kidneys of various groups. Panel C: Densitometric analyses of the Western blotting results. C–F: Each bar graph represents the ratios of p-AMPK to total AMPK (C), Sirt1 to β-actin (D), Ac-H3 to H3 (E) and p66Shc-to-β-actin (F). G–H: The relative associations among pAMPK, Sirt1 and Ac-H3 protein expression: R (p-AMPK/Ac-H3) = −0.811, R(Ac-H3/Sirt1) = −0.895, and R (p-AMPK/Sirt1) = 0.909 (G) and R (Ac-H3/p66Shc) = 0.789, R (Ac-H3/Sirt1) = −0.895, and R (Sirt1/p66Shc) = −0.726 (p

Article Snippet: Other materials, including an In situ BrdU-Red DNA Fragmentation (TUNEL) Assay Kit (ab66110) and anti-β-actin antibody (ab8226), were purchased from Abcam (UK).

Techniques: Expressing, Mouse Assay, Western Blot

Journal: bioRxiv

Article Title: The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods

doi: 10.1101/2020.05.26.116889

Figure Lengend Snippet: Changes in protein levels of autophagy related factors in HG of hamsters in three different photoperiodic groups (a) Representative immunoblots of LC3, P62, and β-actin in three different photoperiodic groups. (b) Ratio of LC3, P62 to β-actin in HG of hamsters in three different photoperiodic groups. Values are means ±SD. n=10. SP, short photoperiod; MP, moderate photoperiod; LP, long photoperiod. Different letters identify statistically significant difference ( P

Article Snippet: The blotted membranes were blocked with 1% BSA in Tris-buffered saline (TBS; 150 mM NaCl, 50 mM Tris-HCl, pH 7.5) and incubated with rabbit anti-bax (1:1000, #50599, Proteintech), rabbit anti-bcl2 (1:1000, #3498, Cell Signaling Technology CST, Danvers, MA, USA), rabbit anti-Cyto C (1:1000, #11940, CST), rabbit anti-LC3 (1:1000), rabbit anti-P62 (1:1000) and rabbit rabbit anti-ATP synthase (1:1000, #14676, Proteintech), rabbit anti-citrate synthase (1:1000, #16131, Proteintech), rabbit anti-DRP1 (1:1000, #12957, Proteintech), rabbit anti-MFF (1:1000, #17090, Proteintech), rabbit anti-FIS1 (1:1000, #10956, Proteintech), and anti-β-actin (1:5000, #20536, Proteintech) in TBS containing 0.1% BSA at 4 °C overnight.

Techniques: Western Blot

Journal: bioRxiv

Article Title: The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods

doi: 10.1101/2020.05.26.116889

Figure Lengend Snippet: Changes in protein levels of mitochondrial related factors in HG of hamsters in three different photoperiodic groups (a) Representative immunoblots of ATP synthase, CS, DRP1, MFF, FIS1, and β-actin in three different photoperiodic groups. (b) Ratio of ATP synthase, CS, DRP1, MFF, FIS1 to β-actin in HG of hamsters in three different photoperiodic groups. Values are means ±SD. n = 10. SP, short photoperiod; MP, moderate photoperiod; LP, long photoperiod. Different letters identify statistically significant difference ( P

Article Snippet: The blotted membranes were blocked with 1% BSA in Tris-buffered saline (TBS; 150 mM NaCl, 50 mM Tris-HCl, pH 7.5) and incubated with rabbit anti-bax (1:1000, #50599, Proteintech), rabbit anti-bcl2 (1:1000, #3498, Cell Signaling Technology CST, Danvers, MA, USA), rabbit anti-Cyto C (1:1000, #11940, CST), rabbit anti-LC3 (1:1000), rabbit anti-P62 (1:1000) and rabbit rabbit anti-ATP synthase (1:1000, #14676, Proteintech), rabbit anti-citrate synthase (1:1000, #16131, Proteintech), rabbit anti-DRP1 (1:1000, #12957, Proteintech), rabbit anti-MFF (1:1000, #17090, Proteintech), rabbit anti-FIS1 (1:1000, #10956, Proteintech), and anti-β-actin (1:5000, #20536, Proteintech) in TBS containing 0.1% BSA at 4 °C overnight.

Techniques: Western Blot

Journal: bioRxiv

Article Title: The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods

doi: 10.1101/2020.05.26.116889

Figure Lengend Snippet: Changes in protein levels of apoptosis related factors in HG of hamsters in three different photoperiodic groups (a) Representative immunoblots of bax, bcl2, Cyto C, and β-actin in three different photoperiodic groups. (b) Ratio of bax, bcl2, Cyto C to β-actin and ratio of bax to bcl2 in HG of hamsters in three different photoperiodic groups. Values are means ±SD. n=10. SP, short photoperiod; MP, moderate photoperiod; LP, long photoperiod. Different letters identify statistically significant difference ( P

Article Snippet: The blotted membranes were blocked with 1% BSA in Tris-buffered saline (TBS; 150 mM NaCl, 50 mM Tris-HCl, pH 7.5) and incubated with rabbit anti-bax (1:1000, #50599, Proteintech), rabbit anti-bcl2 (1:1000, #3498, Cell Signaling Technology CST, Danvers, MA, USA), rabbit anti-Cyto C (1:1000, #11940, CST), rabbit anti-LC3 (1:1000), rabbit anti-P62 (1:1000) and rabbit rabbit anti-ATP synthase (1:1000, #14676, Proteintech), rabbit anti-citrate synthase (1:1000, #16131, Proteintech), rabbit anti-DRP1 (1:1000, #12957, Proteintech), rabbit anti-MFF (1:1000, #17090, Proteintech), rabbit anti-FIS1 (1:1000, #10956, Proteintech), and anti-β-actin (1:5000, #20536, Proteintech) in TBS containing 0.1% BSA at 4 °C overnight.

Techniques: Western Blot

Journal: Retrovirology

Article Title: Human HERC5 restricts an early stage of HIV-1 assembly by a mechanism correlating with the ISGylation of Gag

doi: 10.1186/1742-4690-8-95

Figure Lengend Snippet: HERC5 restricts HIV-1 Gag particle production by a mechanism dependent on its E3 ligase activity . A , U2OS cells were co-transfected with pGag, pUbe1L, pUbcH8, pMyc-ISG15 and either empty plasmid, pHERC5 or pHERC5-C994A. Gag particles released into the supernatant and intracellular Gag protein expression were analyzed after 24 hours by quantitative Western blotting using anti-p24CA and anti-β-actin as a loading control. The same blot was stripped and tested for ISG15 conjugates using anti-myc (bottom blot). B , Cells were co-transfected with empty plasmid or pHERC5 and pUbe1L, pUbcH8, pHis-ISG15 and pGag. Cells were lysed 72 hours later and histidine-tagged proteins were purified using nickel agarose. Purified proteins were resolved using SDS-PAGE and subjected to Western blotting using anti-p24CA. C , Cells were co-transfected with empty vector or pHERC5, pUbe1L, pUbcH8, pHis-ISG15 and pGag with or without pUbp43. Gag particles released into the supernatant and intracellular Gag protein expression were analyzed after 72 hours by quantitative Western blotting using anti-p24CA or anti-β-actin. D , In a similar transfection as in C, histidine-tagged proteins were purified from cell extracts using nickel agarose and subjected to Western blotting using anti-p24CA. The two lanes were digitally separated from the same image. Numerical values on the blots display the densitometric quantification of the specified bands. All Western blots shown are representative of at least two independent experiments.

Article Snippet: Antibodies: anti-HERC5 was obtained from Abnova, anti-FLAG from Sigma, anti-ISG15 and anti-β-actin from Rockland, anti-HA from Roche, anti-myc from Santa Cruz, and anti-ribosomal protein S6 from Cell Signaling Technology.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Purification, SDS Page

Journal: Retrovirology

Article Title: Human HERC5 restricts an early stage of HIV-1 assembly by a mechanism correlating with the ISGylation of Gag

doi: 10.1186/1742-4690-8-95

Figure Lengend Snippet: Intracellular localization of IFNβ-treated HERC5 and HIV-1 Gag . A , Localization of endogenous HERC5 (red) in IFNβ-treated (500 units/ml for 16 hours) PBMCs (scale bar = 5 μm), Jurkat cells (scale bar = 5 μm), and murine dendritic cells (scale bar = 10 μm). Images shown are representative of at least 2 independent experiments. B , Localization of endogenous ribosomes (green) and HERC5 (red) in control Jurkat cells or IFNβ-treated Jurkat cells (scale bar = 5 μm). Localization of Gag alone (red) or HERC5 (green) +/- CS ( C ), or co-expression of Gag and HERC5 + CS ( D ) in U2OS cells 48 hours after transfection. Scale bars = 20 μm. Images shown are representative of at least 3 independent experiments. E , HeLa cells were co-transfected with pR9, pUbe1L, pUbcH8, and pMyc-ISG15 with or without pFlag-HERC5 or pFlag-HERC5-C994A. Gag was immunoprecipitated under non-denaturing conditions using anti-p24CA. Precipitated proteins were resolved using SDS-PAGE and subjected to Western blotting using anti-Flag or anti-p24CA. 10% of the input lysates was subjected to Western blotting using anti-p24CA and anti-β-actin on the same blot. HERC5 protein was undetectable in the 10% input lysates.

Article Snippet: Antibodies: anti-HERC5 was obtained from Abnova, anti-FLAG from Sigma, anti-ISG15 and anti-β-actin from Rockland, anti-HA from Roche, anti-myc from Santa Cruz, and anti-ribosomal protein S6 from Cell Signaling Technology.

Techniques: Expressing, Transfection, Immunoprecipitation, SDS Page, Western Blot

Journal: Retrovirology

Article Title: Human HERC5 restricts an early stage of HIV-1 assembly by a mechanism correlating with the ISGylation of Gag

doi: 10.1186/1742-4690-8-95

Figure Lengend Snippet: HERC5 restricts MLV Gag particle production . U2OS cells were co-transfected with pMLV-Gag and either empty plasmid, pHERC5 + CS, or pHERC5-C994A + CS with or without pUbp43. Gag particles released into the supernatant and intracellular Gag protein expression were analyzed by quantitative Western blotting using anti-MLV antisera or anti-β-actin 48 hours post-transfection. Numerical values on the blots display the densitometric quantification of the specified bands after normalization with β-actin levels.

Article Snippet: Antibodies: anti-HERC5 was obtained from Abnova, anti-FLAG from Sigma, anti-ISG15 and anti-β-actin from Rockland, anti-HA from Roche, anti-myc from Santa Cruz, and anti-ribosomal protein S6 from Cell Signaling Technology.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot

Journal: Retrovirology

Article Title: Human HERC5 restricts an early stage of HIV-1 assembly by a mechanism correlating with the ISGylation of Gag

doi: 10.1186/1742-4690-8-95

Figure Lengend Snippet: HERC5 restricts a late stage of HIV-1 replication . A , Inhibition of HIV-1 particle production. 293T cells were co-transfected with pR9 and either empty plasmid or pHERC5. Twenty-four hours after transfection, infectious HIV-1 virions released into the supernatant were quantified using GHOST(3) indicator cells. B , Inhibition of HIV-1 replication. HOS-CD4/CXCR4 cells were co-transfected with plasmids encoding a replication-competent HIV-1 provirus (R9) and HERC5 or an empty vector control. HIV-1 virions released into the supernatant were pelleted 72 hours after transfection and analyzed by Western blotting using anti-p24CA antibody. p24CA levels were quantified densitometrically. C , Representative Western blot using anti-p24CA of HIV-1 particles released into the supernatant. Data shown are the average of at least two independent experiments performed in triplicate. D-G , 293T cells were co-transfected with pLKO.1/scrambled shRNA or pLKO.1/HERC5 shRNA and pR9. Twenty-four hours after transfection, HIV-1 particles released into the supernatant, cellular RNA and cellular protein were isolated. HERC5, HERC3 and β-actin RNA were detected by reverse transcription polymerase chain reaction (RT-PCR) ( D ) and quantified densitometrically ( E ). HIV-1 particles released into the supernatant and intracellular Pr55Gag protein were detected by Western blotting using anti-p24CA ( F ) and quantified densitometrically ( G ). Data shown are representative of at least two independent experiments.

Article Snippet: Antibodies: anti-HERC5 was obtained from Abnova, anti-FLAG from Sigma, anti-ISG15 and anti-β-actin from Rockland, anti-HA from Roche, anti-myc from Santa Cruz, and anti-ribosomal protein S6 from Cell Signaling Technology.

Techniques: Inhibition, Transfection, Plasmid Preparation, Western Blot, shRNA, Isolation, Reverse Transcription Polymerase Chain Reaction

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

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

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

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

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

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

Journal: Experimental and Therapeutic Medicine

Article Title: Manipulations in HIWI level exerts influence on the proliferation of human non-small cell lung cancer cells

doi: 10.3892/etm.2016.3106

Figure Lengend Snippet: Overexpression of HIWI in non-small cell lung cancer (NSCLC) specimens from 57 patients. (A) Relative mRNA expression levels of HIWI to β-actin in the intra- (n=57) and peri-tumor (n=57) tissues were determined using the reverse transcription-quantitative

Article Snippet: Subsequently, the membranes were incubated with polyclonal rabbit anti-HIWI polyclonal antibody (1:300; 701177; Thermo Fisher Scientific Inc.) or anti-β-actin (1:00; 100162-RP02-100; Sino Biological, Inc., Beijing, China) at 37°C for 1 h, followed by incubation with peroxidase-conjugated secondary antibody (1:500; PA1-096; Pierce Biotechnology, Inc., Rockford, IL, USA) at 37°C for 45 min. Antibody complexes were visualized using the Enhanced Chemiluminescence Detection system (GE Healthcare Life Sciences, Chalfont, UK), according to the manufacturer's protocol.

Techniques: Over Expression, Expressing

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

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

Journal: Scientific Reports

Article Title: Glucocorticoid receptors in the locus coeruleus mediate sleep disorders caused by repeated corticosterone treatment

doi: 10.1038/srep09442

Figure Lengend Snippet: Treatment of 7 days CORT (40 mg/kg, s.c.) decreased GR, but not MR protein levels in the LC. Representative of western blots and quantification of total GR (a), cytoplasm GR (b), nucleus GR (c) and total MR (f) in the LC. (d) The distribution of GR in nucleus/cytoplasm after last injection of CORT. CORT 1D: CORT 40 mg/kg s.c. at day 7 proceeded by 6-day vehicle. (e) β-actin (actin) and histone-3 (histone) are shown as quantitative loading control for cytoplasm and nucleus respectively. Data are represented as mean ± S.E.M. ( n = 6 pooled samples and repeated for 3 ~ 4 times, * p

Article Snippet: The membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies, including anti-β-actin (1:1000; Abmart, Shanghai, China), anti-histone H3 (1:2000; Cell Signaling Technology, MA, USA), anti-GAD (1:1000; Chemicon Millipore, MA, USA), anti-TH (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GR (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-MR (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in TBS-T buffer (Tris-buffered saline +0.1% Tween-20) at 4°C overnight.

Techniques: Western Blot, Injection

Journal: Scientific Reports

Article Title: Glucocorticoid receptors in the locus coeruleus mediate sleep disorders caused by repeated corticosterone treatment

doi: 10.1038/srep09442

Figure Lengend Snippet: Treatment of CORT (40 mg/kg, s.c.) for 7 days increased TH protein levels in the LC and decreased GAD protein levels in the VLPO. Representative of western blots and quantification of TH (a) in the LC and GAD (b) in the VLPO were shown. CORT 1D: CORT 40 mg/kg s.c. at day 7 proceeded by 6-day vehicle. β-actin (actin) is shown as a quantitative loading control. Data are represented as mean ± S.E.M. ( n = 6 pooled samples and repeated for 3 ~ 4 times, * p

Article Snippet: The membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies, including anti-β-actin (1:1000; Abmart, Shanghai, China), anti-histone H3 (1:2000; Cell Signaling Technology, MA, USA), anti-GAD (1:1000; Chemicon Millipore, MA, USA), anti-TH (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GR (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-MR (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in TBS-T buffer (Tris-buffered saline +0.1% Tween-20) at 4°C overnight.

Techniques: Western Blot

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

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

Journal: Science Advances

Article Title: Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth

doi: 10.1126/sciadv.aau9060

Figure Lengend Snippet: GO289 potently and selectively inhibits CK2. ( A to D ) Effect of GO289 on kinase activity in vitro. Activity of CK2 (A) and PIM2 (C) was analyzed in the presence of GO289 at various concentrations ( n = 2). A panel of 60 kinases (B) and DYRK, HIPK, and PIM family kinases (D) were screened with 5 μM GO289 ( n = 2). In (D), the effect of GO289 on multiple kinases is compared to published values for CK2 inhibitors TBB, DMAT, and CX-4945. ND, not determined. ( E ) Effects of CK2 inhibitors on circadian period and reporter signal intensity in Bmal1-dLuc U2OS cells. Changes in period (left) and luminescence intensity (right) compared to the DMSO control are plotted ( n = 4). P values are summarized in table S3. ( F and G ) Effect of GO289 on cellular CK2 activity. HEK293T cells (F) or U2OS cells (G) were treated with GO289 at various concentrations for 24 hours and subjected to immunoblotting with anti-phosphorylated (anti-phospho) CK2 substrate antibody. The membrane was reprobed with anti-phospho PKA (protein kinase A) substrate and anti–β-actin antibodies (F) or stained with CBB (Coomassie Brilliant Blue) (G).

Article Snippet: Protein samples were boiled in SDS sample buffer, separated by SDS-PAGE, and analyzed by immunoblotting with anti-CK2α (Santa Cruz Biotechnology, sc-6479), anti-CK2β (Calbiochem, 218712), anti-phospho CK2 substrate (Cell Signaling Technology, 8738), anti-phospho PKA substrate (Cell Signaling Technology, 9624), and anti–β-actin (Wako Pure Chemical, 017-24573) antibodies.

Techniques: Activity Assay, In Vitro, Staining

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

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

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

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

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

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

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

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

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

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

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

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

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