Journal: PLoS ONE

Article Title: Upregulation of Nuclear Factor-Related Kappa B Suggests a Disorder of Transcriptional Regulation in Minimal Change Nephrotic Syndrome

doi: 10.1371/journal.pone.0030523

Figure Lengend Snippet: A, AP1 DNA-binding activity of 25 microg of nuclear proteins. Nuclear extracts from NFRKB-transfected HEK cells were incubated with the wild-type AP1 oligonucleotide (Wt AP1) or with mutant-type AP1(Mut AP1) oligonucleotide. B, Expression of c-fos and c-jun. Immunoblots were performed with 50 microg of proteins. C, HEK cells were transiently co-transfected with the NFRKB or empty vector (Ev), and AP1-Luc reporter plasmid. The luciferase activity was measured using the “Dual Luciferase reporter Assay”. The data are presented as relative luciferase activity (firefly luciferase/the renilla luciferase). Statistical analyses were carried out on data from five independent experiments using the one-way Anova (** p <0.015; Kruskal-Wallis test).

Article Snippet: The double-stranded oligonucleotide probes (100 ng), with the consensus and mutant NF-kB (sc-2505, sc-2511), GAS/ISRE (sc-2537, sc-2538), NFATc (sc-2577, sc-2578) and AP1 (sc-2501, sc2514) sequences, respectively, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Activity Assay, Transfection, Incubation, Mutagenesis, Expressing, Western Blot, Plasmid Preparation, Luciferase, Reporter Assay

Journal: Oncogenesis

Article Title: Ras-association domain family 10 acts as a novel tumor suppressor through modulating MMP2 in hepatocarcinoma.

doi: 10.1038/oncsis.2016.24

Figure Lengend Snippet: Figure 3. RASSF10 modulated cell cycle. (a) Cell-cycle distribution was analyzed by FACS flow cytometry in QGY7703 cells and HepG2 cells stably transfected with pcDNA3.1-RASSF10 or pcDNA3.1 vector. Restoration of RASSF10 induced the accumulation of HCC cells in G1 cell cycle phase. The asterisk indicates statistical significance (*Po0.05). (b) Western blot shows the expression of major mediators in cell cycle process including p27, CyclinD1, CDK2 and CDK4.

Article Snippet: Primary antibodies used in this study are as follows: RASSF10 (1:1000, catalog number: ab113105), MMP2 (1:1000, catalog number: ab86607) and tissue inhibitor of metalloproteinases 2 (TIMP2) (1:200, catalog number: ab180630) (Abcam, Cambridge, MA, USA); cyclin-dependent kinases2 (CDK2) (1:200, catalog number: sc-748), CDK4 (1:200, catalog number: sc-260), Janus kinase 1/2 (JNK1/2) (1:200, catalog number: sc7345), p38-mitogen activated protein kinase (p38 MAPK) (1:200, catalog number: sc-4708) (Santa Cruz Biotechnology, Dallas, TX, USA); P27(1:1000, catalog number: #3686 s), CyclinD1 (1:1000, catalog number: #2978); extracellular regulated protein kinases (ERK) (1/1000, catalog number: #4695), FAK (1:1000, catalog number: #3285), pFAK397 (1:1000, catalog number: #3283 s) and pFAK 925 (1:1000, catalog number: #3284 s) (Cell Signaling Technology, Beverly, MA, USA); GAPDH (1:1000, catalog number: 85-14- 9523-80) and Tublin (1:1000, catalog number: 85-41-4510-82) (Multisciences Biotech, Hangzhou, China).

Techniques: Cytometry, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: Oncogenesis

Article Title: Ras-association domain family 10 acts as a novel tumor suppressor through modulating MMP2 in hepatocarcinoma.

doi: 10.1038/oncsis.2016.24

Figure Lengend Snippet: Figure 4. RASSF10 retarded tumor growth in vivo. (a) Subcutaneous tumor growth curve of RASSF10-expressing QGY7703 and HepG2 cells in nude mice was compared with vector (pcDNA3.1) transfected cells. The RASSF10 group showed a retarded tumor growth compared with the vector group (HepG2, P = 0.012; OGY7703, Po0.01). The data are means ± s.d. (n = 8/group). (b) A representative picture of tumor growth in nude mice subcutaneously inoculated with RASSF10 or vector (n = 8/group). (c) Histogram represents mean of the tumor weight from the RASSF10 and vector groups. The asterisk indicates statistical significance (*Po0.05, **Po0.01). (d) Cell cycle mediators including p27, Cycling D1, CDK2 and CDK4 were evaluated in the xenograft tumors by RT-PCR.

Article Snippet: Primary antibodies used in this study are as follows: RASSF10 (1:1000, catalog number: ab113105), MMP2 (1:1000, catalog number: ab86607) and tissue inhibitor of metalloproteinases 2 (TIMP2) (1:200, catalog number: ab180630) (Abcam, Cambridge, MA, USA); cyclin-dependent kinases2 (CDK2) (1:200, catalog number: sc-748), CDK4 (1:200, catalog number: sc-260), Janus kinase 1/2 (JNK1/2) (1:200, catalog number: sc7345), p38-mitogen activated protein kinase (p38 MAPK) (1:200, catalog number: sc-4708) (Santa Cruz Biotechnology, Dallas, TX, USA); P27(1:1000, catalog number: #3686 s), CyclinD1 (1:1000, catalog number: #2978); extracellular regulated protein kinases (ERK) (1/1000, catalog number: #4695), FAK (1:1000, catalog number: #3285), pFAK397 (1:1000, catalog number: #3283 s) and pFAK 925 (1:1000, catalog number: #3284 s) (Cell Signaling Technology, Beverly, MA, USA); GAPDH (1:1000, catalog number: 85-14- 9523-80) and Tublin (1:1000, catalog number: 85-41-4510-82) (Multisciences Biotech, Hangzhou, China).

Techniques: In Vivo, Expressing, Plasmid Preparation, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of applied physiology (Bethesda, Md. : 1985)

Article Title: Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men.

doi: 10.1152/japplphysiol.00630.2016

Figure Lengend Snippet: Fig. 3. Basigin antibody validation. Using Western blotting, two duplicate gels (1 and 2) were loaded with four lanes of a single sample of skeletal muscle homogenate (M), six differ- ent concentrations of an internal standard (IS1– IS6: 8–38 g protein), and one lane of a Hep G2 cell lysate positive control (ve). Follow- ing transfer, membrane 1A was immunoblotted with a mouse monoclonal anti-basigin antibody (sc-21746), and membrane 2A was immuno- blotted with a different mouse monoclonal anti- basigin antibody (sc-46700). After imaging, the peroxidase of the horseradish peroxidase- conjugated secondary antibodies was inacti- vated by 15 min of H2O2 incubation. Both membranes were reprobed (1B and 2B) with a goat polyclonal anti-basigin antibody (sc- 9757). See text for additional details on immu- noblotting.

Article Snippet: Primary Rabbit polyclonal anti-MCT1 Merck Millipore AB3540P/2136555 1:1,000 1:15,000 Rabbit polyclonal anti-MCT4 Merck Millipore AB3316P/2397059 1:1,000 1:20,000 Mouse monoclonal anti-basigin Santa Cruz sc-21746/K1913 1:200 1:7,500 Mouse monoclonal anti-NHE1 Merck Millipore MAB3140/2283852 1:500 1:7,500 Rabbit polyclonal anti-NBCe1 Cell Signaling 11867/0001 1:500 1:5,000 Rabbit polyclonal anti-CAII Santa Cruz sc-25596/F0611 1:1,250 1:30,000 Mouse monoclonal anti-CAIII Abnova H00000761-M02/12243-S1 1:2,500 1:40,000 Mouse polyclonal anti-CAIV Abnova H00000762-B02P/08325 WULz 1:500 1:10,000 Mouse polyclonal anti-CAXIV Abnova H00023632-B01P/08358 WULz 1:750 1:10,000 Secondary Goat anti-mouse IgG Perkin Elmer NEF822001EA Goat anti-rabbit IgG Perkin Elmer NEF812001EA J Appl Physiol • doi:10.1152/japplphysiol.00630.2016 • www.jappl.org Overall basigin abundance changed in response to training (time main effect: F3,37.2 4.47, P 0.009) (Fig. 4C).

Techniques: Biomarker Discovery, Western Blot, Positive Control, Membrane, Imaging, Incubation

Journal: Journal of Neuroinflammation

Article Title: Hypoxia-inducible factor-1 (HIF-1) is involved in the regulation of hypoxia-stimulated expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and MCP-5 (Ccl12) in astrocytes

doi: 10.1186/1742-2094-4-12

Figure Lengend Snippet: Effects of hypoxia or CoCl 2 treatment on HIF-1 DNA binding and reporter gene activity in astrocytes. ( A ) Mouse HIF-1α +/+ and HIF-1α +/- cells were exposed to hypoxia or 125 μM CoCl 2 for 6 h, nuclear extracts were then prepared and EMSA carried out as described in the Materials and Methods. HIF-1 in the nuclear extracts isolated from hypoxia or CoCl 2 -treated cells bound to the wild-type probe (lanes #1–6) but not to the mutant probe (lanes #7–12). HIF-1/DNA complex was detected in hypoxia (H)-or CoCl 2 (Co)-treated cells (lanes #2, 3, 5, 6) but not in control (C) cells (lanes #1, 4). More complex (darker band) was seen in hypoxia-or CoCl 2 -treated HIF-1α +/+ cells (lanes #2, 3) than that in hypoxia-or CoCl 2 -treated HIF-1α +/- cells (lanes #5, 6). Supershift assay showed that the HIF-1/DNA complex was shifted up in the presence of wild-type (wt) oligo probe and 4 μg HIF-1α antiboby (lanes #13 & 14). ( B ) The activity of the reporter gene, luciferase yellow, under a wild-type HIF-1-binding sequence from MCP-1 promoter (pGL3/MCP1w) or a mutated sequence (pGL3/MCP1m), was carried out to test the transcriptional activation of MCP-1 by HIF-1 activated by hypoxia. FHAs were transfected with an empty vector, pGL3/MCP1w or pGL3/MCP1m, respectively, and recovered overnight for 16 h. The cells were exposed to normoxia or hypoxia for 4 h and then harvested for luciferase yellow assays. Hypoxia strongly stimulated the reporter gene activity from pGL3/MCP1w but not from the empty vector and pGL3/MCP1m. Each bar represents the mean ± SD of three assays and each assay had at least two replicates. Asterisks indicate significant difference compared to relevant controls (p < 0.05, one-way ANOVA, followed by multiple comparisons among means).

Article Snippet: For the EMSA, a typical double-stranded consensus oligonucleotide for HIF-1 binding (5'-TCTGT ACGTG ACCACACTCACCTC-3') and a mutant DNA sequence (5'-TCTGT AAAAG ACCACACTCACCTC-3') [ , ] were purchased from Santa Cruz Biotech Inc (CAT # sc-2625, Santa Cruz, CA) and end-labeled with γ[ 32 P]-ATP (Mandel/NEN Life Science, Guelph, ON).

Techniques: Binding Assay, Activity Assay, Isolation, Mutagenesis, Control, Luciferase, Sequencing, Activation Assay, Transfection, Plasmid Preparation