For detection of protein-nucleic acid interaction, an electrophoretic mobility shift assay (EMSA) was conducted as described by Hellman and Fried (2007). Breifly, 8-10 μg of nuclear extract protein was incubated with binding reaction-mixture containing either 32P end-labeled double-stranded oligonucleotides (NF-κB, AP-1, p53 or SP-1) or unlabeled oligonucleotide as shown in table 2.3. The reaction mixture was incubated at 37°C for 45-60 min. After completion of reaction, 6μl of 6X DNA-loading dye was added and mixed well by gentle tapping.Table 2.3: Binding conditions for DNA-protein complexes in EMSA

Gel shift assay

The desired S.cerevisiae strain was grown overnight in YPD liquid medium and yeast cells were harvested by centrifugation at 2,500x gin 15 mL polypropylene tubes. Yeast cells were washed with PBS, suspended in 500 μL lysis buffer (Section 2.1.6.2) and were transferred to a 1.5 mL microcentrifuge tube. Yeast cells were incubated for 15 min on a thermomixer set at 65 ̊C and 750 rpm.After incubation, a volume equivalent to 500 μLof glass beads (0.5 mm) were added and cells were lysedon a beating apparatus by mixing three times for 45 seconds each with intermittent cooling on ice to prevent overheating.Cell lysates were centrifuged at 12,000 x gfor 5 min and upper aqueous phase was transferred carefully to a new 1.5 mLmicrocentrifuge tube, to which 275 μL of 3M sodium acetate was added. To this solution, 500 μL of chloroform was added, mixed well, and centrifuged at 12,000 x gfor 5 min at 4°C (this step was repeated twice). Supernatant was transferred to a new 1.5 mLmicrocentrifuge tubeand500 μL of isopropanolwas added and mixed well by inverting the tube 3-4 times. To precipitate genomic DNA, the suspension was centrifuged at 12000 x gfor 15minat 4°C. Precipitated genomic DNA was washed with 70% ethanol and dried at room temperature. The genomic DNA pellet was dissolved either in 100μLof Sigma molecular biology grade water or TE buffer supplied with Qiagen plasmid mini prep kit, andadd 1 μL of RNase solution (30 mg/mL) was added to this and incubated at 37oC for 1 h. The extracted genomic DNA was checked for integrityon a 0.8%agarose gel by electrophoresis and stored at -20oC

Genomic DNA isolation by glass bead lysis method

44KDa to 670 KDa; Ovalbumin (44KDa), Conalbumin (75KDa), Aldolase (158KDa), Ferritin (440KDa), Thyroglobulin (669KDa)] purchased fromGE Healthcare Life Science. Blue dextran was used for the void volume of the column. The molecular weight of the protein complex fractions was calculated from the plot. The different fractions that were collected as eluates from the column were concentrated,and the presence of WWP2 or WWP1 as a monomer or multimer was identified by western blotting using specific antibodies for WWP2 and WWP1

The WWP2-WWP1 heterodimeric complex formation under normal conditions and upon cisplatin-induced stress conditions was studied by gel exclusion chromatography. HEK293T cells untreated or treated with cisplatin were harvested,and cell lysate was prepared by using the standard protocol. Sephacryl S-200 (GE Healthcare) columns were equilibrated with 1X NETN (without Triton X-100) at a flow rate of 1ml/min. 0.8ml of cell lysate (1mg/ml) was passed through the Sephacryl S-200 column,and different fractions (fraction size; 500μl) were collected using Bio-Rad 2110 fraction collectorat the same flowrate. To determine the molecular weight of the fractions, column was calibrated with high molecular weight markers [range

Gel filtration

flash (Thermoscientific). β-Glucuronidase activity for GUS was expressed as nanomoles of MU produced/minute/108 cells

In planta siderophore gene expression was studied by measuring β-glucuronidase activity. GUS marked BXOR1 strain and wild-typeBXOR1 (control) were inoculated in the leaves of 14 day old susceptible Taichung Native 1 (TN-1) variety of rice. After 10 days of inoculation, leaves were crushed and dissolved in 1 ml of MUG extraction buffer without adding MUG substrate (4-methylumbelliferyl β-D-glucuronide). Subsequently, 250 μl extraction buffer containing MUG was added, and incubated at 37°C for appropriate time (Jefferson et al., 1987). Next, 75-μl aliquots were taken from each reaction mixture, and the reaction was terminated by the addition of 675 μl Na2CO3 (0.2 M). Fluorescence was measured against 4-methyl-umbelliferone (MU; Sigma) as standard at excitation/emission wavelength of 365/455 nm, respectively in

In plantaGUS expression assay for siderophore cluster

shRNA were processed for invasion studies. A suspension of 1×105cells in 200μLof 1%FBS containing DMEM was prepared and plated as described in Section 2.2.16. Cells were incubated at 37ºC with 5% CO2for 24 h to allow invasion. Downstream processing of the samples i.e. removing non-invading cells, staining and imaging were followed as described in Section 2.2.16. The number of cells invadedto the lower surface was quantified by counting the total number of DAPI positive nuclei in at least 10 random fields. The total number of cells invaded was normalized to non-targeting control cells and expressed as percentage invasion

Invasion assays were conducted as described previously (Raoet al., 2015).Invasion chambers with pre-coated matrigel (BioCoat Matrigel invasion chamber, 24 well, 8 μm pore size, Corning) were used to conduct the invasion assays. Packages containing invasion chambers were kept outside and allowed to come to room temperature. Inserts were equilibrated by adding with 500 μLwarm (37ºC) 1% FBS containing DMEM to the upper chamber of the insert and bottom well of the companion plate, and rehydrated for 2 h in a humidified tissue culture incubator at 37ºCwith 5% CO2. The rehydration media was removed carefully without disturbing the pre-coated matrigel matrix on the membrane. Meanwhile, HCT116 cells expressing either non-targeting or IP6K1 targeting

Invasion assay

foolish imaginations of his heart

MR. CORNELLIER—All the confederations which you have mentioned were or are republican, and had the common fate of republican institutions. You have not said a word about monarchical confederations. MR. JOLY—I have made no mention of monarchical confederations, because none have ever existed, and none can exist. The principle of a monarchy is that the power resides in one person; the principle of confederation is that it resides in all the members of the confederation. A confederation would, therefore, always be a republic, even if formed of several states subject to a monarchy; because the power would not be vested in one person, but in each of the several states, of which no one would acknowledge a head ; it would be a republic consisting of a very small number of members. Before I take leave of all the confederations, the names of which I have mentioned, I intend to say one word, at least, in their favor. We understand that states

The seat of the Federal Government is to be at Ottawa, of course. The Governor General or other head of this magnificent future vice-royalty, or what not, will hold his court and parliament at Ottawa ; but a handsome sop is thrown to Quebec and Toronto, also. They, too, are each to have a provincial court and legislature and governmental departments.

12

But Theresa May, I think to -- on the heels of the Skripal attack, of the Russian nerve poison on a former agent and for which she garnered considerable international support, riding that wave into this -- and that response was a surprise to her, I think, in many ways, that going into this without getting parliamentary support, which at best would have been ambiguous, given the level of divisiveness and the precariousness of her position in Parliament, meant that she went along with that, without consulting with lawmakers and the backlash has been not just from Jeremy Corbyn or the opposition but also within her party, where there's a lack of consensus on whether or not she should have gone into this with Donald Trump, with French president Emmanuel Macron without greater consultation with the international community.

So, you are talking about a soggy weekend, five, six, 700 upwards of 800 millimeters of rainfall in some of this regions across the northern tier of the island of Kawai.

The attack in Douma was horrific attack on civilians using chemical weapons part of a Civil War that has killed hundreds of thousands of people.

Again after public outrage grew and the two issues as you say, have come together, one is about the continuing concern about violence against women and people are asking more than five years after that awful rape, gang rape in Delhi in 2012.

He ordered a military strike on Syrian chemical weapons facilities but then he hesitated and then reversed direction on advice from his most senior advisers on taking sanctions against companies that helped facilitate that deadly chemical weapons infrastructure by Syria.

Well in everything any time that you're talking about Russia and the relationship between the United States and Russia, it's always in the context of this cloud that's over the president about whether or not he's got some kind of ulterior motive for his policies and looked the combination of James Comey going out there over the last several days saying I don't know if the Russians have something that they are using to blackmail the president, and then the sort of abrupt, like hey, I don't want to do sanctions on Russia, you know, yes, there were some sanctions but he was very much pushed in the direction by Capitol Hill and others.

$130,000, if that's viewed as a campaign contribution, that is $127,300 above the legal limit.

It's $131,000.

This was back in 2013 President Obama looked at that chemical attack, and at that point in time, his general was saying, hey, it's not a slam-dunk, because you can detect there were Syrian claims, you can detect Syrians dropped bombs, but it's sometimes hard to know if they dropped an existing vehicle of weapons, and Syria has been known to use chemical weapons, or if the Syrians did it.

I will do two things, balance the budget, balances over five years with a 1 percent across the board cut, but also expand health savings accounts larger than anyone has tried in the past.

A 1 percent cut each year is about $13 billion, actually balances the budget in five years.

I think when you win by three million votes but lose by less than 80,000 votes in three states that decided the election, there's a lot that should be factors, but I think this was a very significant factor.

They are originally from bacteria in fungi and they were designed to be much faster, 10 hundred times faster than the original within a period of five to 10 years.

But I'll tell you, some of these flexes that we're having here, one story I picked up that I've never told before or written, but William Webster, former head of the CIA, told me that -- it's one of his best stories, he's allowing me to tell it -- that they created a body double for George Herbert Walker Bush at CIA, meaning somebody who looked just like him and it was uncanny how much CIA had created a human that looked like 41.

Do you -- do you -- do you -- I'm sorry, do you disagree with the $4 million for school safety -- $4 billion for school safety, the $6 billion for opioids