Reproductive Biology and Endocrinology 2008, 6:49

Western blot analysis Cells were washed with ice-cold PBS and lysed by cold lysis buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS in PBS, pH 7.4), including freshly added protease inhibitor (Sigma, St. Lois, MO). The extracts were placed on ice for 10 min, vortexed briefly and centrifuged for 15 min at 4°C to remove cell debris. The total protein concentration was determined using a Bradford assay (Bio-Rad Laboratories, Mississauga, ON). The samples were boiled for 10 min before running the gels. SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a 10% separating gel. Twenty-five to 30 μg of protein was then electrotransferred to a nitrocellulose membrane (Bio-Rad Laboratories). The membrane was blocked with 5% skim milk in PBS. PBX2 and actin were detected by primary antibodies in 5% skim milk/TBS for 1 hr at 25°C. Subsequently, the signals were detected with secondary antibodies conjugated with horse radish peroxidase and visualized by ECL (Pierce, Rockford, IL).

MMP ZymographyBlood plasma samples were taken from the delayed 6-hour tPA only group and the combination minocycline plus tPA group. Samples were collected before ischemia, before tPA administration, 1 hour after tPA, and 24 hours after ischemia. Standard gel zymography was used to measure levels of MMP-2 and MMP-9.5 EDTA blood samples were immediately centrifuged at 4000 rpm for 15 minutes to obtain supernatants and total protein concentrations determined with the BCA assay (Pierce). Thirty micrograms of total protein were loaded and separated by a 10% Tris-glycine gel with 0.1% gelatin as substrate, washed with renaturing buffer (Invitrogen) for 90 minutes and further incubated with developing buffer (Invitrogen) at 37°C for 24 hours. Finally, the gels were stained with 0.5% Coomassie blue R-250 for 1 hour and then destained appropriately. MMP-2 and MMP-9 human standards (same molecular weight as rat proteins) were loaded in each gel to identify the bands and to standardize between gels. Band intensities of total MMP-9 and MMP-2 (including pro- and cleaved forms) were quantified via standard gel densitometry techniques as previously described5,11 and expressed as a percentage of pro-MMP-9 standards in each gel. This calculation using a common known protein amount as the baseline denominator allowed quantitation and comparisons between all gels.

J. Clin. Invest. 118(1): 124-132 (2007).

Southern blot analysis and PCR genotyping. DNA sequences for PCR genotyping primers are as follows: 5′-TTCTGCAGACCATGAACTGATCACTAGT-3′; 5′-TAGAGCAAGTACCAGAATAGCCAGG-3′; and 5′-ACAGGAAAGGACAATACAAGAAAGCAC-3′. Southern blot probes were generated by PCR using the following primer sets: 5′ probe forward, 5′-AAGCTTAGAATTTACAGCCTGCAGCGCTGA-3', reverse, 5′-GGATCCGAGCTAAGGTTTTGTTAAGTTTCC-3′; and 3′ probe forward, 5′-CTCGAGTCATGCATCCTGTGTGAA-3′; reverse, 5′-GGATGACTTTGTTTAGGCTGTTGC-3'. In brief, tail genomic DNA was digested with EcoRI and analyzed using a standard Southern blot protocol. The 25 μl PCR genotyping reaction contained 1 μl of tail DNA as template, 2.5 U of Taq polymerase (Promega) in 0.5 μl, and a final concentration of 2 mM MgCl2. An annealing temperature of 70°C was used and the PCR products were electrophoresed on a 2% agarose gel.

J. Clin. Invest. 118(1): 79-88 (2007)

Immunoblotting analyses. MACS-sorted B cells and MEFs were lysed in RIPA buffer and proteins quantified using a BCA assay (Pierce Biotechnology). Proteins (25 or 50 mg/lane) were electrophoretically separated on 4%–12% SDS-PAGE gels (Invitrogen), transferred to membranes (Protran; Schleicher and Schuell), and blotted with antibodies specific for β-actin, Puma (Sigma-Aldrich), c-Myc, p62 (Santa Cruz Biotechnology Inc.), p53 (BD Biosciences — Pharmingen; IC12, Cell Signaling), p19Arf (kindly provided by Charles Sherr, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA), cathepsin D (R&D Systems), and LC3 (polyclonal antibody raised in rabbits). Following incubation with primary antibodies, the blots were then incubated with appropriate anti-mouse, anti-rat, or anti-rabbit immunoglobulin secondary antibodies (Amersham). Bound immunocomplexes were detected by enhanced chemiluminescence (Amersham) or ECL SuperSignal (Pierce Biotechnology).

J. Clin. Invest. 118(1): 64-78 (2007)

The receptor tyrosine kinase EphA2 promotes mammary adenocarcinoma tumorigenesis and metastatic progression in mice by amplifying ErbB2 signalingJ. Clin. Invest. Dana M. Brantley-Sieders, et al. 118:64 doi:10.1172/JCI33154 [Go to this article.] Figure 3Elevated EphA2 expression in MCF10A. ER2 cells enhances cell proliferation and invasiveness in vitro. (A) Parental MCF10A human breast cells and MCF10A.HER2 cells were transduced with adenoviruses (Ad) expressing EphA2 or control β-gal and plated on growth factor–reduced Matrigel to generate 3-dimensional spheroid cultures. After 10 days in culture, parental MCF10A cells and cells expressing Ad–β-gal formed small, round acinar structures, while MCF10A.HER2 cells formed larger colonies with irregular, invasive protrusions (arrows). Expression of Ad-EphA2 in MCF10A cells resulted in larger, irregular colonies, an effect that was amplified in MCF10A.HER2 cells (P < 0.05; single-factor ANOVA). Scale bar: 25 μm. (B) Cultures were stained with TO-PRO-3 iodide nuclear stain (red) and anti-Ki67 (green) and imaged by confocal microscopy. Confocal analysis revealed that parental and Ad–β-gal–transduced MCF10A formed uniform acinar structures composed of a single layer of epithelial cells surrounding a central lumen, while MCF10A.HER2 cells formed multiacinar structures with invasive protrusions (arrows) and a poorly defined lumen containing several cells. MCF10A cells transduced with Ad-EphA2 also formed multiacinar structures with a poorly defined lumen. Invasion and lumen filling were enhanced in MCF10A.HER2 cells overexpressing EphA2. Scale bar: 20 μm. EphA2 overexpression significantly enhanced proliferation (Ki67+ nuclei, arrows) within acinar structures formed by MCF10A and MCF10A.HER2 cells (P < 0.05; single-factor ANOVA). (C) Expression of adenoviral gene products and overexpression of ErbB2/HER2 in MCF10A.HER2 cells was confirmed by immunoblot, and uniform loading was verified by immunoblot for actin. Expression of p-Erk, total Erk, p-EphA2, and total EphA2 was also assessed by immunoblot.

J. Clin. Invest. 118(1): 51-63 (2007)

Nucleic acid hybridization and probes. Total RNA isolation and Northern hybridization were performed as described previously (57). Blots containing 5–10 μg of RNA from snap-frozen mammary glands and tumors were hybridized to cDNA probes generated by RT-PCR. The primer pairs were as follows: β-actin, forward 5′-TGAGACCTTCAACACCCCAG-3′ and reverse 5′-TGAGACCTTCAACACCCCAG-3′; Axin2, forward 5′-CCGAGCTCATCTCCAGGC-3′ and reverse 5′-GGACAGAGGCAGCGGACTC-3′; Cyclin D1, forward 5′-ACCCTGACACCAATCTCCTCAAC-3′ and reverse 5′-TGCTTGTTCTCATCCGCCTCTG-3′; Lef1, forward 5′-CCCATCATATGATTCCTGG-3′ and reverse 5′-GTCGACTCCTGTAGCTTC-3ι; Mitf, forward 5′-ATACAGTCACTACCAGGTGCAGACC-3′ and reverse 5′-CAAGTTTCCAGAGACGGGTAACG-3′; Snail, forward 5′-GTGTGGAGTTCACCTTCCA-3′ and reverse 5′-TATCTCTTCACATCCA-3′; Sox10, forward 5′-TGTGTGCCCTGCTCCTCATCAG-3′ and reverse 5′-GGCAGCGATGTGTTACATGTGG-3′. After subcloning, the identity of each probe was confirmed by DNA sequence analysis. Southern hybridization to detect p53 and Ink4a/Arf alleles were performed as described previously (27, 31).

J. Biol. Chem., Vol. 282, Issue 52, 37567-37574

FIGURE 3.Disrupting hsp90 protein-protein interactions with eNOS inhibits ACh-induced vasodilation of facialis arteries in vitro and ex vivo but not NO-donor DETA-NONOate-induced vasodilation. A, the SB2 peptide (6.2 ?FONT size=-2>M) disrupts eNOS interactions with hsp90 in lysates from proliferating BAEC. IP, immunoprecipitates; WB, Western blot. B, acute exposure (10 min) of isolated, pressurized facialis arteries to TSB2 (2.9 ?FONT size=-2>M) markedly reduces ACh-dependent vasodilation compared with untreated vessels (p < 0.02, n = 6?0), whereas treatment with TSB(Ctr) control peptide with Glu to Ala substitutions had no effect on vasodilation. C, 10 min exposure of isolated, pressurized facialis arteries to TSB2 (2.9 ?FONT size=-2>M) has no effect on DETA-NONOate-induced vasodilation compared with 2.9 ?FONT size=-2>M TSBSCR-treated vessels (n = 4). D, C57BL/6 mice were treated with TSB2 (1 mg/kg) or PBS (100 祃) for 2 weeks. At the end of this treatment period facialis arteries from TSB2-treated and untreated C57BL/6 mice were isolated, pressurized, and examined for responses to ACh as previously described (9). This line graph shows that TSB2 treatments alter vascular responses to ACh. Not only is ACh-induced vasodilation reduced, but other mechanisms of vasodilation are developing in vessels from TSB2-treated mice based on the fact that L-NAME fails to reduce vasodilation to base line.

The Journal of Neuroscience, April 16, 2008, 28(16):4172-4182

Immunoprecipitation and immunoblotting. Immunoprecipitation was performed to study phosphorylation of 18 kDa subunit of complex I, Fe-S subunit of complex III, and cytochrome oxidase IV (COX IV) subunit of complex IV phosphorylation in different experimental conditions. The total synaptosomal fraction (500 礸) was dissolved with 0.3% SDS, sonicated two times for 5 s each, and then immediately adjusted to 0.1% of the SDS concentration in "immunobuffer" consisting of 50 mM Tris-HCl, 1% Triton X-100, 1% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and 0.5% NP-40, pH 7.4, and incubated for 20 min at room temperature. The immunoprecipitation was performed using protein A-Sepharose beads (Sigma-Aldrich) and anti-phospho-threonine, anti-phospho-serine, and anti-phospho-tyrosine mouse monoclonal antibodies (Sigma-Aldrich) per the manufacturer's instructions. The samples were washed twice with immunobuffer after overnight incubation of samples with protein A-Sepharose beads (Sigma-Aldrich) and anti-phospho-threonine, anti-phospho-serine, or anti-phospho-tyrosine. The resulting pellet was used for immunoblotting using anti-18 kDa subunit of complex I, anti-Fe-S subunit of complex III, or anti-COX IV subunit of complex IV antibody. To determine the levels of PKC, PKC, PKC, cytochrome c, synaptophysin, and ?actin, 40 礸 of protein from either synaptosomal or soluble fraction was separated using 12% PAGE. The immunoblotting was performed using anti-PKC, anti-PKC, anti-PKC, anti-cytochrome c, anti-synaptophysin, and/or anti-?actin antibodies. Immunoreactivity was detected using enhanced chemiluminescence (ECL Western blotting detection kit; GE Healthcare, Little Chalfont, Buckinghamshire, UK). Western blot images were digitized at eight-bit precision by means of a CCD-based camera (8?2 bits) (Xillix Technologies, Vancouver, British Columbia, Canada) equipped with a 55 mm Micro-Nikkor lens (Nikon, Tokyo, Japan). The camera was interfaced to an image acquisition and analysis system (MCID model M2; Imaging Research, St. Catherines, Ontario, Canada). The digitized immunoblots were subjected to densitometric analysis using MCID software.

The Journal of Neuroscience, December 5, 2007, 27(49):13446-13456

Figure 1. Development of spontaneous synaptic activity in rat cortical cultures correlates with surface and synaptic trafficking of AMPA receptor subunits GluR1 and GluR2. A, Representative traces of spontaneous synaptic activity in sister cultures of rat E18 cortical neurons at subsequent DIV, measured by whole-cell voltage clamp at a holding potential of –65 mV. Network-driven spontaneous synaptic activity develops slowly in culture, increasing dramatically after the first week in vitro, with a rapid increase in unitary event amplitude and frequency between 10 and 14 DIV. B, Live labeling the surface pool of GluR1 and GluR2 subunits in these cultures revealed a strong increase in the surface expression of AMPAR subunits between 6 and 14 DIV. Data shown are example fields of view for cultures live labeled with anti-GluR1 (top images) and anti-GluR2 (middle images) antibodies and merged images with the dendritic marker MAP2 (bottom images) at 6,10, and 14 DIV. C, Surface biotinylation and Western blot analysis of receptors in culture at increasing days in vitro confirms the surface immunofluorescence data showing the increase in GluR1 and GluR2 surface expression over this time period. D, Cumulative data from multiple cultures are presented showing the increase in synaptic localization of these GluR puncta measured by costaining with an excitatory synaptic marker (VGlut1 and VGlut2) (mean ± SEM).

Reproductive Biology and Endocrinology 2008, 6:44

Western blot analysis Western blot analysis was performed as described previously [20]. Briefly, cells were grown in 60 mm dishes and treated with vehicle (control), PDBu and IFNT (50 ng and 1000 ng), alone and in combination for 6 h as described above. Cells were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% SDS, 0.5% sodium deoxycholate, 1 mM DTT, 100 μM PMSF, 1% NP-40), and 1 × protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Lysates from triplicate treatment wells were combined, sonicated and centrifuged (10,000 g, 20 minutes) and supernatants were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Non-specific binding sites were blocked with 5% non-fat dry milk and membranes were incubated with antibodies to PLA2G4A, PLA2G4C, and PLA2G6 in 1:800, 1:400 and 1:1000 dilutions, respectively, overnight at 4°C. Membranes were rinsed 3 times and protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences, Arlington Heights, IL). A blot was prepared from each of the replicated experiments.

Reproductive Biology and Endocrinology 2008, 6:49

Effects of FSH and GDF-9 on PBX2 in SVOG cells. The cells were treated with FSH (100 ng/ml) or/and GDF-9 (50 or 100 mg/ml). For co-treatment, cells were pre-treated with FSH for 4 hr and then treated with GDF-9 for 24 hr. Ota et al. Reproductive Biology and Endocrinology 2008 6:49 doi:10.1186/1477-7827-6-49

PLoS Med. 2006 October; 3(10): e420.

Figure 4 Status of KEAP1 and NRF2 Is Altered in Cancer Cells (A) Immunoblot showing increased nuclear localization of NRF2 in nuclear extracts (NE) from cancer cells. Cancer cells showed lower levels of KEAP1 (~69 kDa) and higher levels of NRF2 (~110 kDa) in total protein lysates (TP). NIVT and KIVT indicate NRF2 and KEAP1 in vitro transcribed/translated product, respectively. (B and C) Quantification of NRF2 and KEAP1 protein in immunoblots. For band densitometry, bands in nuclear extract blot (B) were normalized to Lamin B1, and those in total protein (C) were normalized to GAPDH. (D) Heat map showing relative expression of KEAP1, NRF2, and NRF2-dependent genes by real-time RT-PCR. Raw data for the heat maps are presented in Table S5.

Reproductive Biology and Endocrinology 2005, 3:37

Southern analysis of the AR locus. Stickleback genomic DNA (20 μg), from one male (lanes 1–3) and one female (lanes 4–6) kidney, digested with EcoRI (lane 1 and 4), SacI (lane 2 and 5) and BamHI (lane 3 and 6). The position of molecular weight markers (kb) is given in the right margin. Olsson et al. Reproductive Biology and Endocrinology 2005 3:37 doi:10.1186/1477-7827-3-37

Reproductive Biology and Endocrinology 2005, 3:37

Southern analyses of stickleback genomic DNA Southern analyses were performed using 20 レg aliquots of male or female stickleback genomic DNA digested with 10 units of either SacI, BamHI or EcoRI at 37∑C for 8 h according to Sambrook et al. [16]. Membranes were probed using [メ32P]-dCTP radiolabelled partial cDNA encoding stickleback AR, which was isolated and sequenced as described above. Hybridizations were performed at 60∑C O/N in 5 】 SSC, 0.02% SDS (w/v), 0,1% N-laurylsarcosine (w/v) and 1% blocking solution (w/v) (Roche). Membranes were washed for 2 】 30 min periods at 42∑C and 60∑C in 0.1 】 SSC, 0.1% (w/v) SDS. Membranes were exposed to Hyperfilm⑩-MP and were visualized as described above.

Am J Pathol. 2008 July; 173(1): 68–76.

Figure 3 Egr-1 regulates genes involved in gliosis. A: Egr-1 loss-of-function effects on human astrocytes transfected with siRNAs against Egr-1. RT-PCR analysis shows that knockdown of Egr-1 leads to reduction in the expression levels of genes encoding ECM components of the glial scar. c, control mock-transfected cells; −, Egr-1 knockdown. DNA size markers are shown on the left. Gene name abbreviations are as follows: CSPG2, 3, 4, chondroitin sulfate proteoglycan 2, 3, 4, respectively; Lamα1, laminin α1; Lamα2: laminin α2; Lamβ1: laminin β1. Real-time PCR quantification of the effects of the Egr-1 knockdown on the expression of putative gene targets shows a 60% drop in phosphacan RNA levels in Egr-1 siRNA-treated astrocytes. Adjusted for aldolase, relative phosphacan expression was 12.96 ± 1.21 U in controls versus 5.05 ± 0.89 U after Egr-1 knockdown (n = 5, P < 0.001). B–E: Immunofluorescence analysis after Egr-1 overexpression in astrocytes transfected with the CMV-Egr-1-IRES-EGFP construct. Transfected, EGFP-positive cells (green, marked by arrows), stain more intensely with antibodies recognizing laminin α1 (B, red) and phosphacan (C, red, anti-RPTPβ) than nontransfected neighboring cells. No difference in expression levels of GFAP (D, red) or β-tubulin (E, red) between Egr-1-overexpressing cells (green, marked by arrows) and nontransfected cells. Superimposed images (far right) confirm that transfected cells express higher levels of putative Egr-1 targets, but similar levels of other proteins. F: Western blotting of proteins isolated from astrocytes transfected with siRNAs against Egr-1 (Egr-1 siRNA), control siRNA (scrambled siRNA), or mock-transfected cells (c). siRNAs against Egr-1 diminish effectively Egr-1 protein levels and lead to down-regulation of phosphacan (Pcan, detected with the KAF13 antibody). β-Tubulin (β-Tub) levels remain unaffected serving as control. Quantification of blot images shows that Egr-1 protein levels are reduced 3.0-fold versus control (mock-transfected cells; SD, 0.4) and 2.8-fold (SD, 0.2) versus scrambled siRNA-transfected cells; phosphacan protein levels are down-regulated 2.18-fold versus control (mock; SD 0.1) and 2.23 times (SD 0.1) versus scrambled siRNA. G: Western blotting of proteins isolated from astrocytes transfected with the CMV-Egr-1-IRES-EGFP (Egr-1 cDNA) construct or the empty vector (c). Egr-1 protein levels increase 2.08-fold (SD, 0.4) leading to 1.42-fold up-regulation of phosphacan (Pcan; SD, 0.14). β-Tubulin (β-Tub) serves as control. Am J Pathol. 2008 July; 173(1): 77–92.