Notes: The inner membrane protein 4-amino-4-deoxy-L-arabinose transferase (ArnT) catalyzes the final step in the polymyxin-resistance pathway in S. typhimurium and Escherichia coli. The authors expressed and purified ArnT as a His₆-tagged protein in E. coli. Briefly, 2 liters of cells expressing ArnT were grown overnight and induced for 3 hours with 1mM IPTG. A French press and pressure cell were used to lyse cells, and the membrane fraction was pelleted using a high-speed centrifugation. Membrane proteins were extracted using 1% dodecylmaltoside, followed by another high-speed centrifugation. The solubilized membrane fraction was passed through a 3ml Q-Sepharose® column. The flowthrough was incubated with 1ml of HisLink™ Protein Purification Resin overnight with gentle mixing. The HisLink™ Resin was washed with a wash buffer containing 10mM imidazole, and the ArnT protein was eluted in a two-step elution using an elution buffer with 100mM and 200mM imidazole. The Q-Sepharose® column was added to the protein purification scheme to eliminate the copurification of E. coli elongation factor Ef-Tu, which contains a cluster of histidine residues and can bind to the HisLink™ Resin if not previously removed. (3854)

Notes: Few reliable prognostic markers have been developed for breast cancer. Human tissue kallikreins (hKs) are serine proteases with diverse functions. Fifteen human tissue kallikrein genes (KLK) have been identified. Several human tissue kallikreins have been associated with malignancies, such as hK3 (prostate-specific antigen) and prostate cancer. hK7 has been linked to a significantly poorer prognosis for ovarian and breast cancer, indicating that it may serve as a marker for these diseases. However, previous analyses of KLK7 expression were non-selective studies of all KLK7 mRNA forms. The authors developed a quantitative reverse-transcription-PCR (QPCR) assay, using the Reverse Transcription System on RNA, then developed a highly sensitive QPCR assay, to determine whether a more specific analysis of full-length KLK7 mRNA might lead to similar results as those from previous studies on the multiple KLK7 mRNA forms, with the ultimate goal of determining whether KLK7 could serve as a marker for breast cancer. (3615)

Notes: The Kinase-Glo^® Luminescent Kinase Assay was used in 384-well plates to test a library of 720 peptides, potential substrates for protein kinase A. In addition, various concentrations of kinase 1 and 3 were tested in 384-well and 1536-well plates, and the results determined using the Kinase-Glo^® Assay. (3728)

Notes: A panel of 11 Gram-negative and 11 Gram-positive bacterial species was used to develop a real-time PCR detection method. Initially DNA was purified from 1ml of various dilutions of bacteria resuspended in a saline solution using either the QIAamp DNA blood mini kit or the Wizard^® SV Genomic DNA Purification System. The results suggested that the Wizard^® SV Genomic DNA Purification System extraction protocol was superior in disrupting the bacterial cell wall (especially of Gram-positive bacteria), to allow release of bacterial DNA. Using DNA purified with the Wizard^® SV Genomic DNA Purification System, detection of S. aureus and E. coli at concentrations as low as 10¹ CFU per PCR was achieved. The Wizard^® SV Genomic DNA Purification System purification protocol provided in eNotes online (www.promega.com/enotes/applications/ap0051_tabs.htm) was used with the following modifications: The bacterial pellet was resuspended in 400µl of enzymatic lysis solution (47mM EDTA, 25mg/ml lysozyme, 20µg/ml lysostaphin) and incubated for 2 hours at 37°C. Next, 19.2mg/ml proteinase K was added (final concentration 0.4mg/ml), and the mixture was incubated for 1 hour at 55°C. Finally, the Nuclei Lysis Solution and RNase Solution were added, mixed and incubated for 10 minutes at 80°C. For real-time PCR analysis, 2µl of genomic DNA was used. (3676)

Notes: In this study, GoTaq^® DNA Polymerase was used in amplification reactions to test for the recruitment of DNA damage checkpoint proteins RPA, Rad9, and ATR in ChIP assays. DNA was isolated from cells treated with UV irradiation or acetoxyacetylaminofluorene. The DNA and DNA damage checkpoint proteins RPA, Rad9, and ATR were crosslinked together and the complexes immunoprecipitated with antibodies toward RPA, Rad9, and ATR. Amplification reactions contained primers designed to amplify regions of the p53 and β-globin genes. (3366)

Notes: Leishmania donovani infection elicits an immune response in mice macrophages that includes the upregulation of nitric oxide synthase 2 (NOS2). Hamster and human macrophages do not exhibit an upregulation of NOS2 upon infection. The authors measured the activities of the NOS2 promoter in response to interferon-γ (IFNγ) and lipopolysaccharide treatment of mouse, hamster and human macrophages. The mouse, hamster and human NOS2 promoters were cloned into pGL3-Basic Vector and transfected into mouse macrophages by electroporation. Promoter activities were determined using the Dual Luciferase^® Reporter Assay System. The pRL-null Vector was used to normalize for differences in transfection efficiency (3470)

Notes: TRAP220/MED1 is amplified in estrogen receptor-positive breast cancer cells and has been shown to interact with a number of transcription factors essential for cell growth and development including BRCA-1 and p53. TRAP220/MED1 is a subunit of the TRAP/Mediator coactivator complex. These authors used RNA interference to reduce TRAP220/MED1 expression by >90%, then microarray analysis to identify genes that were downregulated after TRAP220/MED1 depletion. One such gene was Aurora-A serine/threonine kinase. The authors created Aurora-A-firefly luciferase constructs to determine the effect of TRAP220/MED1 depletion on Aurora-A promoter activity. As a positive control, the authors used a thyroid hormone (T₃)-responsive firefly luciferase construct to show that depletion of TRAP220/MED1, which is known to play a role in nuclear receptor-mediated gene activation, interferes with thyroid hormone receptor-mediated activation of T₃-responsive genes. Luciferase reporter gene activity was measured using the Dual Luciferase Reporter Assay System, and results were normalized to Renilla luciferase expression from the pRL-TK Vector. (3607)

Notes: This study investigated whether prequalene diphosphate (PSDP) is an endogenous regulator of phosphatidyl 3-kinase (PI3K). The Kinase-Glo^® Luminescent Kinase Assay was used to determine the activity of recombinant human p110gamma-PI3K activity. A total of 4pmol of PI3K/reaction was exposed to PDSP or a PI3K inhibitor followed by 25µg L-α-phosphatidylinositol and 1µM ATP before adding the Kinase-Glo^® Reagent. (3546)

Notes: Heat-shock transcription factor 1 (HSF1) induces the expression of heat-shock proteins upon activation by heat or other stress events. This report shows that activation of HSF1 is mediated by a ribonucleoprotein complex containing the translation elongation factor eEF1A and a noncoding RNA termed heat-shock RNA1 (HSR1). An antisense oligonucleotide and siRNA constructs directed against HSR1 were used to suppress expression in BHK cells. The viability of these cells was then evaluated after heat-shock using the CellTiter^® 96 AQueous Non-Radioactive Cell Viability Assay. (3399)

Notes: The authors wanted to develop a more sensitive assay to detect codon 816 pathogenic variations in people diagnosed with systemic mastocytosis. The Wizard^® Genomic DNA Purification Kit was used to isolate DNA from peripheral blood and bone marrow aspirate samples. To extract DNA from 2–5 micron, paraffin-embedded samples of bone marrow trephine, skin, spleen or liver, the tissues were digested with Proteinase K for four days at 56°C prior to DNA purification using the Magnesil^® Genomic Fixed Tissue System. The isolated DNA was subjected to two assays: enriched sequencing of mutant alleles (ESMA) after BsmAI restriction enzyme digestion, and allele-specific competitive blocker PCR (ACB-PCR). (3575)

Notes: Three alleles were identified in the promoter of the serotonin transporter, HTTLPR, based on the number of a specific repeat sequence and a single nucleotide polymorphism found in the repeat sequence. Sequences from these alleles (named S, L_A, and L_G) were cloned into the pGL4.10 [luc2] Vector. These constructs were then tested by transfecting them into RN46A cells derived from rat dorsal raphe neurons. The constructs containing the L_G and S allele sequences displayed a 2.8 fold increase in activity compared to the construct containing the L_A allele sequence. Data was represented as relative luciferase levels compared to a control (pGL4.10 [luc2] Vector). (3411)

Notes: To test the ability of SorICD to stimulate transcription of a Gal4-dependent luciferase promoter, COS-7 cells were transfected with pG5luc, pBIND, or pBINDSorICD from the CheckMate™ Mammalian Two-Hybrid System. Luciferase expression was analyzed using the Bright-Glo™ Luciferase Assay System. (3376)

Notes: To examine the effect of Substance P (SP) on COX-2 expression, the COX-2 promoter region spanning –2069 to –66 bp was cloned by PCR and subcloned into the pGL3-Basic Vector (pGL3-Cox-2). Nontransformed human colonic epithelial NCM460 cells overexpressing neurokinin-1 receptor (NK-1R; NCM460-NK-1R) were seeded in 12-well plates and transiently transfected with pGL3-Cox-2 with either a transfection control pRL-TK Vector or siRNA or both. The siRNA molecules used were for AK1, JAK2 (Upstate Biotechnology), STAT3, STAT5, STAT6 or a control siRNA. The transfected cells were serum starved for 24 hours, treated with SP for 4 hours and then lysed. The cell extracts were measured for firefly and Renilla luciferase activities using the Dual-Luciferase^® Reporter Assay System. Relative luciferase activity was a ratio of COX-2 promoter firefly activity to Renilla activity; results were expressed as percentage of control group without SP stimulation. To mutate the STAT binding elements, the pGL3-Cox-2 construct was modified using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The resulting mutant constructs were tested in the same system as the wildtype COX-2 promoter. (3520)

Notes: The COX-2 promoter was cloned by PCR into the pGL3 Vector. NCM460-NK-1R cells were transiently transfected with the cloned promoter and the pRL-TK vector as an internal control or siRNA targeted against various JAK/STAT genes. The Dual-Luciferase^® Assay was used to measure promoter activity. The wildtype COX-2 promoter was mutagenized using the GeneEditor™ in vitro Site-Directed Mutagenesis System. (3384)

Notes: To examine the putative role that SUN1, a nuclear envelope (NE) protein with a C-terminal SUN (Sad1/UNC-84 homology) domain, may play in the inner nuclear membrane, the murine SUN1 cDNA was subcloned into the pCI-neo Mammalian Expression Vector. Primers containing the myc or HA tag sequence were used to amplify the pCI-neo construct, adding tags both 5’ and 3’ of the SUN1 cDNA. Emerin, a marker for the NE lumen, was also subcloned into the pCI-neo Vector and a 3’ myc tag added. These constructs were translated and labeled with ³⁵S in vitro using the TNT^® T7 Quick Coupled Transcription/Translation System, and the radiolabeled proteins were used in a GST pull-down assay. In addition, NIH/3T3 cells were transiently transfected with myc-SUN1 constructs, fixed in methanol and then co-stained with anti-myc and anti-lamin A/C antibodies. (3511)

Notes: The transcription factors BMAL1 and CLOCK form a heterodimer that positively regulates the Period (Per) and Cryptochrome (Cry) genes, which are involved in maintenance of circadian oscillations in mammals. The PER and CRY proteins negatively regulate the activity of the BMAL1/CLOCK heterodimer. To investigate the specific role of BMAL1 in maintenance of circadian rhythms, Per or Bmal1 promoter-driven luciferase reporter vectors were created using the pGL4.11[luc2P] luciferase reporter vector. Forty-eight hours after transfection of Rat-1 fibroblasts with each reporter construct, cellular clocks were synchronized by treatment with 100nM dexamethasone, and rhythmic expression of the luciferase reporter was observed. The effect of various mutant BMAL1 constructs on luciferase expression was then evaluated using this model system. (3472)

Notes: The authors of this study investigate the relationship between BRCA1 and activity of the progesterone receptor (PR) in mammary tumor cells. BRCA1 mutations confer increased risk for steroid hormone-responsive cancers such as endometrial and cervical cancers and prostate cancer. In this study, evidence for interaction between PR and BRCA1 is presented. Glutathione-S-transferase (GST) capture assays were used to determine if PR and BRCA1 interact directly. GST-PR fusion proteins are used to pull down in vitro transcribed and translated BRCA1. In vitro transcription and translation reactions were carried out using a TNT^® Rabbit Reticulocyte Lysate system. Interaction between BRCA1 and PR isoforms A and B was observed, and this interaction did not appear to require the presence of progesterone. The authors also showed that BRCA1 regulates expression of several progesterone-responsive genes. (3602)

Notes: The Protein S promoter (PROS1) fragment –5948/–1 was cloned directly 5’ to the firefly luciferase reporter gene in the pGL3-Basic Vector using the KpnI and XhoI enzyme sites. This construct, PS5948-luc, was linearized with KpnI and NdeI and subsequently subjected to progressive deletion. The size of the resulting 5’-deletion was determined by sequence analysis, and the deletion constructs were used for transient transfection assays. HepG2, HuH7, HeLa and HUVEC cells were transfected at 60–80% confluency in 12-well plates using 3µl of Tfx™-20 per microgram DNA. In each transfection, an equimolar concentration of construct was used and supplemented with an additional plasmid to keep the amount of transfected DNA constant. pRL-SV40 Vector was co-transfected as a transfection control using a 1:500 ratio to the total transfected amount of DNA in HepG2, HuH7 and HeLa cell lines, and a 1:100 ratio in transfections with HUVEC and 1 × 10⁶ Meg01 suspension cells. Transcription factor expression vector (250ng) was co-transfected, and expression vector without the transcription factor cDNA was used as a negative control. Cell extracts were harvested at either 24 (HepG2 and HuH7) or 48 hours (Meg01, HUVEC, and HeLa) post-transfection using 250µl of Passive Lysis Buffer per well. Luciferase activity was determined using 20–100µl of lysate with the Dual-Luciferase^® Reporter Assay System. (3510)

Notes: This study investigated the role of phosphatases in the deactivation of the Cystic Fibrosis transmembrane conductance regulator (CFTR). CFTR C-terminal peptides were synthesized and immobilized. They were incubated with recombinant PP2A B´ε subunit produced from Rabbit Reticulocyte Lysate or BL21 cells. The recombinant B´ε subunit binds specifically to CFTR-(1451-1476) C-terminal peptide. The core PP2A subunit and A regulatory subunit do not bind to CFTR. (3528)

Notes: Monocarboxylate transporters (MCT) transport lactic acid across the cell membrane. The promoters of 4 MCT family members (MCT1, MCT2, MCT3 and MCT4), were amplified by PCR and cloned into the pGEM^®-T Easy Vector. The sequences were confirmed, and the promoters were cloned into the pGL3-Basic Vector. The Dual-Glo™ Luciferase Assay System was used to quantitate promoter activity under basal and hypoxic conditions in HeLa cells. The pRL-SV40 Vector was used to normalize for differences in transfection efficiency. (3463)

Notes: In this study, an in vitro assay was performed to examine the ribonuclease activity of the herpes simplex virus virion host shutoff (vhs) protein. To test whether a purified GST-vhs fusion protein exhibited RNase activity, the entire 3’ UTR of human IEX-1 mRNA (a known target of vhs) was labeled with ³²P and then incubated at 30°C with either GST-vhs or GST alone. Samples were taken at various time points and analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. The GST-vhs fusion protein displayed RNase activity. When RNasin^® Plus RNase Inhibitor was added to the reaction, no degradation products were seen, confirming that the nuclease activity of GST-vhs could be blocked by an RNase inhibitor. (3391)

Notes: These authors characterized the interaction between whirlin, a protein component of stereocilia in the inner ear, and the membrane-associated guanylate kinase protein p55. Portions of p55 and whirlin were expressed in the TNT^® T7 Coupled Transcription/Translation System as ³⁵S-labeled protein. GST-fusion proteins of the guanylate kinase of p55, the PDZ3 domain of whirlin or GST alone were expressed in E. coli. Interaction between the ³⁵S-labeled proteins and the GST-fusion proteins were examined in GST pull-down assays using the MagneGST^® Pull-Down System. GST alone was used as a negative control. Briefly, the MagneGST^® particles were washed eight times with wash/binding buffer supplemented with 0.05% Nonidet^® P-40, and bound proteins were resuspended in 2X SDS sample buffer and analyzed on 12.5% SDS polyacrylamide gels. (3683)

Notes: In this study, the Caspase-Glo^® 8 and Caspase-Glo^® 9 Assays were used to assess caspase activation in Zebrafish embryos after radiation exposure. After irradiation, caspase activation, morphological abnormalities, and DNA fragmentation were observed, all three of which could be partially reversed by treatment with the radiomodifier amifostine. The Caspase-Glo^® 9 Assay was shown to be sensitive enough to detect the effects of radiation on as few as 2 embryos. The authors concluded that the Caspase-Glo^® Assays provided an effective and convenient means for rapidly assessing the lethal effects of radiation on Zebrafish embryos, and for assaying the ability of the radiomodifier to counteract these effects. The assay could be performed within hours of radiation exposure directly on the embryos in a 96-well plate format. (3571)

Notes: While exploring existing breast cancer cDNA microarrays the authors noted that alpha-basic-crystallin (αB-crystallin) was frequently expressed in basal-type breast carcinomas and wondered if αB-crystallin might contribute to the aggressive nature of these types of breast cancer. To identify how αB-crystallin disrupts mammary acinar architecture, the analyzed MCF-10A cell pools, grown in 1% horse serum (no added EGF) for activation of signaling pathways key to cell transformation. They used the CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay to examine cell viability at various days, compared to day 0. The authors observed that MCF-10A-αB-WT cells expressed higher levels of total and phosphorylated ERK1/2, Akt and p38 than did non-αB-containing parental cell types. (3614)

Notes: Uses CellTiter 96 Aqueous One Solution for cell viability determination (3313)