rabbit anti goat type iv collagen  (SBA Inc)

Journal: PLoS Neglected Tropical Diseases

Article Title: Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

doi: 10.1371/journal.pntd.0000727

Figure Lengend Snippet: The cryosections were obtained 15 minutes after i. d. injection of 10 µg of Alexa488-Jar and 50 µg Alexa488-BnP1 in mouse skin. Samples injected with Alexa488-BSA were used as a control. The blood vessels were stained with anti-rat CD-31 polyclonal antibody (red). Alexa488-Jar accumulated in the venules walls in the hypodermis region (B-green) as well as on the basement membrane of skeletal muscle cells and capillaries (E). Alexa488-BSA (A, D) and Alexa488-BnP1 (C, F) did not concentrate around these structures. The sections were examined with a Confocal Microscope LSM 510 Meta (Zeiss).

Article Snippet: Then, the sections were incubated with donkey anti-rat CD-31 polyclonal antibody (BD Bioscience, USA), at a 1∶40 dilution, or goat anti-rabbit type IV collagen polyclonal antibody (Chemicon, USA), at a 1∶40 dilution, for 18 hours at 4°C.

Techniques: Injection, Staining, Microscopy

Journal:

Article Title: Angiotensin II Induces Renal Plasminogen Activator Inhibitor-1 and Cyclooxygenase-2 Expression Post-Transcriptionally via Activation of the mRNA-Stabilizing Factor Human-Antigen R

doi: 10.2353/ajpath.2009.080652

Figure Lengend Snippet: AngII induces a nucleo-cytoplasmic HuR shuttling and a subsequent increase in HuR binding to COX-2 and PAI-1 mRNAs in vivo. A: Representative Western blot analysis showing AngII-induced changes in the cytoplasmic HuR content in whole kidney homogenates. Rats were infused for the indicated time periods with vehicle (−) or with AngII (+) (400 ng/kg/minute). Whole kidney homogenates from three animals of each group were pooled and prepared for cell fractionation. Thirty μg of cytoplasmic fractions or 10 μg of nuclear extracts were subsequently subjected to SDS-PAGE and immunoblotted with an anti-HuR-specific antibody. To correct for variations in the protein loading, blots were stripped and successively incubated with anti-β-actin and anti-histone deacetylase 1 (HDAC1) antibodies, respectively. B: Pull-down assay from cytoplasmic fractions (300 μg) of tissue homogenates from rats treated for 14 days with either vehicle or with AngII (400 ng/kg b.w.), which were immunoprecipitated with 2 μg of anti-HuR (a.-HuR) or with the same amount of mouse IgG (IgG). The RNA-bound by HuR was harvested and subjected to qRT-PCR by using either COX-2- or PAI-1-specific primers as indicated from the coding regions of the corresponding genes. Amounts of input RNA added to the IP reaction mixture was normalized by assessment of GAPDH levels from input RNA by RT-PCR (not shown). Data show the results of a pull-down experiment from cytoplasmic extracts originating from a pool of three animals (per group) and are depicted as -fold induction compared with vehicle-treated animals.

Article Snippet: Antibodies raised against β-actin, collagen-type IV, COX-2, HDAC1, HuR, PAI-1, anti-goat, anti-rabbit, and anti-mouse horseradish peroxidase-linked IgGs, were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Binding Assay, In Vivo, Western Blot, Cell Fractionation, SDS Page, Incubation, Histone Deacetylase Assay, Pull Down Assay, Immunoprecipitation, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Angiotensin II Induces Renal Plasminogen Activator Inhibitor-1 and Cyclooxygenase-2 Expression Post-Transcriptionally via Activation of the mRNA-Stabilizing Factor Human-Antigen R

doi: 10.2353/ajpath.2009.080652

Figure Lengend Snippet: The increase in HuR shuttling by AngII is accompanied by an up-regulation of COX-2 and PAI-1 in vivo. A: Corresponding to the increase in HuR-bound mRNAs, AngII infusion causes an increase in the total steady-state levels of COX-2 and PAI-1 mRNA as determined by qRT-PCR and normalizes COX-2 and PAI-1 mRNA contents to GAPDH mRNA level. Data represent means ± SD (n = 3) and are presented as -fold induction compared with vehicle (*P ≤ 0.05, **P ≤ 0.01). B: Whole kidney homogenates from rats that had been infused for 14 days with either vehicle or with AngII were fractionated for assessment of cytoplasmic HuR (cytoplasmic), total HuR, or PAI-1 and COX-2 contents (total), respectively. Thirty μg of each fraction were subjected to Western blot analysis and cytoplasmic fractions were successively probed with anti-HuR- and with anti-β-actin-specific antibodies. Total protein extracts were additionally probed with an anti-PAI-1- and anti-COX-2-specific antibody. The Western blots shown indicate an up-regulation of cytoplasmic HuR (top) in three individual animals corresponding with an increase in the total COX-2 and PAI-1 levels. Bottom: Summary of total COX-2, PAI-1, cytoplasmic, as well as nuclear HuR protein levels in rats treated for 14 days with either vehicle or with AngII (400 ng/kg b.w.). To correct for variations in the protein loading, blots were stripped and successively incubated with anti-β-actin and anti-HDAC1 antibodies, respectively. Results are means ± SD (n = 3) and are presented as induction versus vehicle-treated control animals (*P ≤ 0.05, ***P ≤ 0.005).

Article Snippet: Antibodies raised against β-actin, collagen-type IV, COX-2, HDAC1, HuR, PAI-1, anti-goat, anti-rabbit, and anti-mouse horseradish peroxidase-linked IgGs, were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: In Vivo, Quantitative RT-PCR, Western Blot, Incubation, Control

Journal:

Article Title: Angiotensin II Induces Renal Plasminogen Activator Inhibitor-1 and Cyclooxygenase-2 Expression Post-Transcriptionally via Activation of the mRNA-Stabilizing Factor Human-Antigen R

doi: 10.2353/ajpath.2009.080652

Figure Lengend Snippet: AngII induces cytoplasmic accumulation of HuR and nuclear import of PKC-δ. A: Representative Western blot showing cytoplasmic (top), nuclear (middle), and total (bottom) HuR levels after treatment with different pharmacological inhibitors. MCs were serum-starved for 16 hours and subsequently remained untreated (−) or were stimulated for an additional 2 hours with AngII (+) (100 nmol/L) in the absence (vehicle) or presence of either valsartan (100 nmol/L), CGP42112 (100 nmol/L), staurosporine (100 nmol/L), or rottlerin (10 μmol/L) as indicated. All inhibitors were preincubated for 30 minutes before the addition of AngII. Protein lysates from cytoplasmic (20 μg), nuclear (10 μg), or whole cell extracts (30 μg) were subjected to SDS-PAGE and immunoblotted with an anti-HuR-specific antibody. To correct for variations in the protein loading, blots were successively incubated with anti-β-actin and anti-HDAC1 antibodies, respectively. B: Top: AngII increases cytoplasmic HuR levels in vivo by an AT1-dependent mechanism. Rats were infused for 6 hours with vehicle (−) or with AngII (+) (400 ng/kg/minute) in the absence (+ vehicle) or presence of either valsartan (20 mg/kg/day), or PD123319 (30 mg/kg/day) or, alternatively, with each inhibitor alone. Whole kidney homogenates from three animals of each group were pooled and prepared for cell fractionation. Thirty μg of cytoplasmic fractions were subsequently subjected to SDS-PAGE and immunoblotted with an anti-HuR-specific antibody. To correct for variations in the protein loading, blots were stripped and successively incubated with an anti-β-actin antibody. PGE2 levels in the corresponding tissue homogenates are depicted in the bottom panel with the data representing means ± SD (n = 3). *P ≤ 0.05 compared with animals treated for 6 hours with vehicle, or #P ≤ 0.05 versus AngII-treated animals. C: AngII promotes nuclear translocation of PKC-δ. Time course of PKC-δ entry into the nucleus by AngII. Quiescent MCs were treated with vehicle (5 minutes, 120 minutes) or with AngII (100 nmol/L) for the indicated time periods before cells were lysed for nuclear and cytoplasmic extracts. Nuclear (50 μg) or cytoplasmic (20 μg) extracts were subjected to SDS-PAGE and immunoblotted with an anti-PKC-δ-specific antibody. To ascertain equal protein contents within the extracts, the blots were stripped and reprobed with an anti-β-actin and with a HDAC1-specific antibody. The Western blots shown are representative of two independent experiments with similar results.

Article Snippet: Antibodies raised against β-actin, collagen-type IV, COX-2, HDAC1, HuR, PAI-1, anti-goat, anti-rabbit, and anti-mouse horseradish peroxidase-linked IgGs, were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Western Blot, SDS Page, Incubation, In Vivo, Cell Fractionation, Translocation Assay