Journal: BMC Cardiovascular Disorders

Article Title: The rat STSL locus: characterization, chromosomal assignment, and genetic variations in sitosterolemic hypertensive rats

doi: 10.1186/1471-2261-3-4

Figure Lengend Snippet: Alternative splicing of Abcg8 mRNA expression in SD, SHR and WKY rats by RT-PCR Panel A shows the gene organisation of Abcg8 , and below it the organisation of an alternatively spliced form detected by sequencing clones from a rat intestinal cDNA library (see Text). Panel B shows that the effect of the inclusion of exon 2b in the mRNA results in an in-frame addition of 22 amino acids. Total RNAs were prepared from liver and small intestine from SD, SHR and WKY rats maintained on a standard rodent chow diet and analysed by RT-PCR with primers (see Methods) based outside the alternatively spliced exon 2b. Direct amplification of a pooled cDNA library sample indicated a major DNA product of 396 bp, with a minor product of 462 bp, containing exon 2b. DNA products, using cloned wild type Abcg8 cDNA (wt) and alternatively spliced cDNA (AS) are shown in tracks 3 and 4 respectively. As can be seen, liver and intestine RNA from all three strains show a minor DNA product of 462 bp (confirmed by sequencing) contained the insertion of exon2b sequences.

Article Snippet: Rat cDNAs for Abcg5 and Abcg8 were identified previously by amplification of these sequences from a Sprague Dawley rat enterocyte cDNA library, using primers located in areas conserved between mouse and human (Genbank AF312714 and AF351785 respectively).

Techniques: Alternative Splicing, Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Clone Assay, cDNA Library Assay, Amplification

Journal: BMC Cardiovascular Disorders

Article Title: The rat STSL locus: characterization, chromosomal assignment, and genetic variations in sitosterolemic hypertensive rats

doi: 10.1186/1471-2261-3-4

Figure Lengend Snippet: Protein Expression of Abcg5 in liver total membrane preparations. Membrane preparations (20 μg protein) from rat tissues were isolated and analysed by SDS-PAGE, blotted and probed with anti- Abcg5 antiserum (R116, panel A), probed with anti-transferrin antiserum (panel B) or the gel was fixed and stained with Coomassie Brilliant Blue R-250 (panel C). Lane 1 contains a recombinant E. coli expressed N-terminal fragment of sterolin-1 as a control for antibody binding, lanes 2 and 3 contain Sprague-Dawley (SD) spleen membrane extracts. Liver from Wistar (lanes 4 and 5), SHR (lanes 6 and 7) and Sprague-Dawley (SD, lanes 8 and 9) Densitometry measurements when normalised to transferrin show a 2-fold increase in sterolin-1 protein in SHR rat liver samples, compared to SD and Wistar rat liver. No antibody for sterolin-2 is currently available.

Article Snippet: Rat cDNAs for Abcg5 and Abcg8 were identified previously by amplification of these sequences from a Sprague Dawley rat enterocyte cDNA library, using primers located in areas conserved between mouse and human (Genbank AF312714 and AF351785 respectively).

Techniques: Expressing, Membrane, Isolation, SDS Page, Staining, Recombinant, Control, Binding Assay

Journal: Nature Communications

Article Title: A Vaccinia -based system for directed evolution of GPCRs in mammalian cells

doi: 10.1038/s41467-023-37191-8

Figure Lengend Snippet: a Comparison of the populations after selection with PTH’(1–34)-HL647 (left panel) or M-PTH(1–14)-HL647 (right panel) to wild-type PTH1R: sort 1 (red line), sort 2 (blue line), sort 3a (black line), sort 3b (green line, repetition of 3a), negative control (dark gray shaded). b Expression analysis of 43 evolved PTH1R variants assessed in live HEK293T cells by flow cytometry analysis with saturating concentrations of PTH’(1–34)-HL647. Expression levels are relative to wild-type receptor expression and are given as mean values ± s.e.m. of 2–3 independent experiments (Supplementary Table ). c cAMP accumulation of 43 evolved PTH1R variants after stimulation with 1 µM PTH(1–34). Data represent maximal cAMP concentrations relative to PTH1R. Bars represent mean values ± s.e.m. of 3–6 independent experiments performed in duplicates (Supplementary Table ). d Ligand affinities of of 43 evolved PTH1R variants in comparison to PTH1R. IC 50 values were derived from whole-cell ligand competition-binding experiments with M-PTH(1–14) or PTH(1–34). Bars represent the mean change ± s.e.m. in calculated affinity (∆pIC 50 ) for each mutant compared with wild-type receptor from 2–8 independent experiments performed in duplicates (Supplementary Table ). b – d Ligands used for selection are indicated below the bar plots. Source data are provided as a Source Data file.

Article Snippet: cDNA libraries for rat NTR1 and human PTH1R were custom-synthesized by MorphoSys AG as linear DNA fragments using the Slonomics® technology , , .

Techniques: Comparison, Selection, Negative Control, Expressing, Flow Cytometry, Derivative Assay, Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: A Vaccinia -based system for directed evolution of GPCRs in mammalian cells

doi: 10.1038/s41467-023-37191-8

Figure Lengend Snippet: a Evolved PTH1R variants exhibit high-affinity agonist binding but equal or reduced affinity for partial or inverse agonists in cells. Competition ligand binding curves of full [M-PTH(1–14) and PTH(1–34)], partial [PTH(3–34)] and inverse agonist [IA-PTH(7–34)], measured in whole cells expressing wild-type PTH1R or 5 evolved variants. b High agonist affinity of evolved PTH1R variants is similar to that of wild-type PTH1R in the G protein-bound state. Ligand binding curves of M-PTH(1–14) to evolved PTH1Rs were measured in membrane fractions, obtained from cells expressing PTH1R wild-type or 5 evolved variants, in the absence (left panel) or presence (middle panel) of 12.5 µM mini-G s . Relative binding constants obtained by competition of M-PTH(1–14) binding with labeled M-PTH(1–14), measured in whole cells (from a ) vs. membrane fractions in the absence or presence of 12.5 µM mini-G s (right panel). Positive values thus reflect stronger binding on membrane fractions than on cells. c Increased agonist affinity is G protein-dependent. Binding of 40 nM M-PTH(1–14) was measured in membrane fractions supplemented with increasing concentrations of mini-G s . Data are relative to binding levels of PTH1R in the absence of mini-G s (gray area). Left panel: Variants showing an increased basal and G protein-dependent increase in ligand binding. EC 50 values are indicated as dotted vertical lines (PTH1R: 1.39 ± 0.2 µM, P34_05: 0.28 ± 0.06 µM, P34_13: 0.38 ± 0.1 µM). Right panel: Variants showing an increase in ligand binding independent of G protein. d G protein-independent increase in ligand binding is due to higher basal activity. cAMP levels were measured 30 min after addition of the phosphodiesterase inhibitor IBMX to cells expressing PTH1R variants and were normalized to receptor expression levels. Data represent mean values ± s.e.m. of 3 experiments performed in duplicates. ** p = 0.0044, **** p < 0.0001. Statistical significance was determined by one-way ANOVA and Bonferroni multi-comparison. Source data are provided as a Source Data file.

Article Snippet: cDNA libraries for rat NTR1 and human PTH1R were custom-synthesized by MorphoSys AG as linear DNA fragments using the Slonomics® technology , , .

Techniques: Binding Assay, Ligand Binding Assay, Expressing, Membrane, Labeling, Activity Assay, Comparison