Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins-1

<p><b>Copyright information:</b></p><p>Taken from "Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins"</p><p>http://www.biomedcentral.com/1472-6750/7/45</p><p>BMC Biotechnology 2007;7():45-45.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1950093.</p><p></p>DS and BatchPrimer were two PERL programs used for extraction of the DNA coding sequence from a full-length sequence (ORF) and design of gene specific primers

Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins-0

<p><b>Copyright information:</b></p><p>Taken from "Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins"</p><p>http://www.biomedcentral.com/1472-6750/7/45</p><p>BMC Biotechnology 2007;7():45-45.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1950093.</p><p></p>nd at 94°C for denaturation, 30 second for annealing, 140 second at 68°C for extension and 10 minutes at 68°C for final extension. Annealing temperature was variable: it started from a relatively high temperature (55°C), and then decreased 1–2 degree each time until to 46°C. The temperature again increased 5 degree and stabilized at 51°C

Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins-8

<p><b>Copyright information:</b></p><p>Taken from "Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins"</p><p>http://www.biomedcentral.com/1472-6750/7/45</p><p>BMC Biotechnology 2007;7():45-45.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1950093.</p><p></p

Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins-5

<p><b>Copyright information:</b></p><p>Taken from "Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins"</p><p>http://www.biomedcentral.com/1472-6750/7/45</p><p>BMC Biotechnology 2007;7():45-45.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1950093.</p><p></p>2 (NP_496422), B2 corresponds to the well showed in Figure 6 and Table 3, and NP_496422 is the accession number of the protein in the public database [40]. The bands labeled with ''Cut'' in the figure correspond to the results after the cleavage by the thrombin and those labeled with ''Uncut'' correspond to the results before the cleavage. ''Aa'' in the figure stands for the amino acid range of the purified proteins

Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins-2

<p><b>Copyright information:</b></p><p>Taken from "Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins"</p><p>http://www.biomedcentral.com/1472-6750/7/45</p><p>BMC Biotechnology 2007;7():45-45.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1950093.</p><p></p

Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins-7

<p><b>Copyright information:</b></p><p>Taken from "Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins"</p><p>http://www.biomedcentral.com/1472-6750/7/45</p><p>BMC Biotechnology 2007;7():45-45.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1950093.</p><p></p>DS and BatchPrimer were two PERL programs used for extraction of the DNA coding sequence from a full-length sequence (ORF) and design of gene specific primers

The α_2BAR dependent hypertensive response is diminished in spinophilin deficient mice.

<p>(A) Mean arterial pressure (MAP) measured in Sp^+/+ and Sp^-/- mice in the same genetic background after UK14,304 injection (0.1mg/kg i.v.). (B) Quantitation of agonist-induced changes in MAP(ΔMAP) over the basal level. (C) Quantitation of area under curve of the hypertensive response curve. n = 5 for each group. *, <i>p</i> <0.05, Sp^+/+<i>vs</i>. Sp^-/-.</p

Del301-303 α_2BAR has a stronger interaction with spinophilin than WT α_2BAR.

<p>(A) Cells co-expressing Myc-spinophilin together with HA-tagged WT or Del301-303 α_2BAR were stimulated with 100μM epinephrine (plus 1μM propranolol to block βARs). (B) Quantitation of the agonist-induced fold change of Myc-spinophilin in the IP complex with the WT α_2B or Del301-303 α_2BAR. n = 3–5 for each condition. **, <i>p<</i>0.01, WT <i>vs</i>. Del301-303 α_2B.</p

Overexpression of spinophilin aa151-444 sufficiently attenuates α_2BAR phosphorylation.

<p>(A) HEK293 cells co-expressing HA-α_2BAR t.ogether with or without Myc-Sp151-444 were stimulated with 100μM epinephrine (plus 1μM propranolol to block βARs) for indicated time points. (B) Quantitation of agonist-induced fold change in α_2BAR phosphorylation. Data were mean ± SEM. n = 4 for each condition. **, <i>p</i><0.01; ***, <i>p</i><0.001 by unpaired Student <i>t</i> test, Myc-Sp151-444 <i>vs</i>. vector control. (C) Overexpression of Sp151-444 showed no effect on α_2BAR phosphorylation in CosM6 cells, which have a low level of endogenous arrestin expression. CosM6 cells co-expressing HA-α_2BAR together with or without Sp151-444 were stimulated. Representative blots from multiple independent experiments are shown.</p

The Del301-303 α_2BAR shows impaired interaction with arrestin 3.

<p>(A) Agonist treatment failed to promote interaction of the Del301-303 α_2BAR with arrestin 3. Cells co-expressing GFP-tagged arrestin3 (GFP-Arr3) with HA-tagged WT α_2BAR or Del301-303 α_2BAR were stimulated with 100μM epinephrine (plus 1μM propranolol to block βARs), and the interaction between arrestin and either WT or Del301-303 α_2BAR was examined by co-IP assays. (B) Quantitation of the agonist-induced fold change of GFP-Arr3 in the IP complex with the WT or Del301-303 α_2BAR. n = 3–4 for each condition. **, <i>p<</i>0.01, WT <i>vs</i>. Del301-303. (C) Del 301–303 α_2BAR was unable to interact with constitutively active mutant arrestin3 R170E following agonist stimulation. Cells co-expressing GFP-Arr3R170E together with WT or Del301-303 α_2BAR were stimulated with 100μM epinephrine (plus 1μM propranolol). (D) Quantitation of the agonist-induced fold change of GFP-Arr3R170E in the IP complex with the WT or Del301-303 α_2BAR. n = 3–4 for each condition. *, <i>p<</i>0.05, WT <i>vs</i>. Del301-303.</p