Journal: Neuron

Article Title: Age-dependent effects of apoE reduction using antisense oligonucleotides in a model of β-amyloidosis

doi: 10.1016/j.neuron.2017.11.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level was used as a reference (Mm99999915_g1 Gapdh).

Techniques: Purification, Virus, Recombinant, Bicinchoninic Acid Protein Assay, Sequencing, Control, Software

Journal: STAR Protocols

Article Title: Cloning BRD4 long isoform into overexpression vectors for stable overexpression of BRD4-L in mammalian cells

doi: 10.1016/j.xpro.2022.101785

Figure Lengend Snippet:

Article Snippet: Download the appropriate sequences. a. Download the Reference Sequence for Homo sapiens bromodomain containing 4 (BRD4), transcript variant long, mRNA (GenBank: NM_058243.3) from the NCBI’s website, using the GenBank data base ( BRD4-L RefSeq ). b. Download the sequence for LentiV_Blast (Addgene, cat# 111887) from Addgene’s website ( LentiV_Blast ).

Techniques: Virus, Recombinant, Cloning, Gel Extraction, Plasmid Preparation, Sequencing, Over Expression, Expressing, Software, Imaging

Journal: PLoS Genetics

Article Title: UVB Induces a Genome-Wide Acting Negative Regulatory Mechanism That Operates at the Level of Transcription Initiation in Human Cells

doi: 10.1371/journal.pgen.1004483

Figure Lengend Snippet: ChIP-seq was carried out using the anti-Pol II antibody (N-20) on human MCF7 cells 1–6 hours after UVB irradiation and on non-treated cells and the corresponding data sets were generated. Average Pol II profiles were calculated for every refseq gene from −1 kb region in front of every transcription start site (TSS), including the gene bodies (GBs), until +4 kb downstream from the 3′ end of annotated genes (EAGs) for all the time points (A) and −1 kb and +1 kb the around TSSs of every refseq gene (B). TSSs (in A and B) and EAGs (in A) are labeled. Pol II ChIP-seq signals are represented in blue and the mock ChIP-seq signals in red. The Y axis shows mean tag densities. Data sets obtained from irradiated cells harvested at the indicated time points in hour (h) after UVB irradiation together with the non-irradiated (Control) cells are represented.

Article Snippet: The MCF7 human cell line (obtained from American Type Culture Collection; reference number HTB-22) was grown in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% foetal calf serum (FCS).

Techniques: ChIP-sequencing, Irradiation, Generated, Labeling, Control

Journal: PLoS Genetics

Article Title: UVB Induces a Genome-Wide Acting Negative Regulatory Mechanism That Operates at the Level of Transcription Initiation in Human Cells

doi: 10.1371/journal.pgen.1004483

Figure Lengend Snippet: Representation of Pol II read number changes obtained by ChIP-seq on MCF7 cell populations following UVB irradiation (as indicated) on the GB regions of the selected 4500 expressed genes when compared to that of the control sample. Pol II tag numbers were calculated on the GB (−100 bp from TSS until EAG) of the selected genes and represented in log 10 value on the y-axis. On the x axis, 4500 expressed genes are sorted based on their genomic lengths labeled in base pairs. Genes are classified from longest genes (from left) to shortest genes (at the right). Blue dots indicate tag numbers at genes in the control sample, and red dots indicate tag numbers at genes in samples harvested (A) 1 hour (h), (B) 2 h, (C) 3 h, (D) 4 h, (E) 5 h and (F) 6 h following UVB irradiation. See also for the total quantifications of the read numbers obtained in the different samples with the corresponding statistical tests.

Article Snippet: The MCF7 human cell line (obtained from American Type Culture Collection; reference number HTB-22) was grown in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% foetal calf serum (FCS).

Techniques: ChIP-sequencing, Irradiation, Control, Labeling

Journal: PLoS Genetics

Article Title: UVB Induces a Genome-Wide Acting Negative Regulatory Mechanism That Operates at the Level of Transcription Initiation in Human Cells

doi: 10.1371/journal.pgen.1004483

Figure Lengend Snippet: From the ChIP-seq datasets heat maps were generated to follow Pol II behavior in time on the 4500 expressed genes at different genomic regions after UVB treatment in MCF7 cells. Tag numbers were calculated and visualized around the TSS (−/+300 bp), on the gene body (−100 bp from TSS until EAG) and downstream from EAG (from EAG to EAG+4 kb) of each gene. To be able to uniformly follow and represent the diverse Pol II occupancy changes on thousands of genes, the calculated Pol II reads at the different regions of different genes (as indicated) were converted into percentage values, where the control non-UVB treated values represent 100% reads (black color code) and the Pol II read alterations are either represented as % loss (green color) or as % gain (red color code). Each horizontal line represents one gene. The intensity of the color on the heatmap represents the magnitude of the Pol II tag number alteration (max −/+73%). Black color also refers to the lack of Pol II density change. In addition, genes were sorted into distinct groups and subgroups (Group Aa-Ag and Group B) based on the Pol II tag density values and patterns by using k-means clustering. Different time points following UVB irradiation are labeled at the top of the panels in hour (h). C = control, non-irradiated data set. For individual genes see .

Article Snippet: The MCF7 human cell line (obtained from American Type Culture Collection; reference number HTB-22) was grown in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% foetal calf serum (FCS).

Techniques: ChIP-sequencing, Generated, Control, Irradiation, Labeling

Journal: PLoS Genetics

Article Title: UVB Induces a Genome-Wide Acting Negative Regulatory Mechanism That Operates at the Level of Transcription Initiation in Human Cells

doi: 10.1371/journal.pgen.1004483

Figure Lengend Snippet: MCF7 cells were transfected with either scrambled siRNA or siRNAs targeting CSB (ERCC6) (see also ), 72 hours following siRNA transfection cells were treated with UVB, or not. ChIP was carried out 3 hours following UVB treatment of MCF7 cells as well as on non-treated cells. Q-PCRs were carried out to monitor Pol II (dark blue bars) (in A), and p62 (light blue bars) (in B) occupancy on the promoters of genes selected from group A ( ubc, rplp1 ), group B ( p21, wdr24 ) and on a negative control ( intergenic ) region in the presence or absence of CSB, as indicated. Control ChIPs (NoAb, red bars) was carried out with Sepharose G beads only. The occupancy values at the promoters are represented in input %. Error bars represent +/− standard deviations in two biological replicates.

Article Snippet: The MCF7 human cell line (obtained from American Type Culture Collection; reference number HTB-22) was grown in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% foetal calf serum (FCS).

Techniques: Transfection, Negative Control, Control

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Optimization of pGC-A expression in Sf9 cells. Western blot (anti-pGC-A) of total cell protein over different virus multiplicities of infection (MOI) and transfection time. MOI of 0 indicates no virus transfection. pGC-A (red arrow) is approximately 120 kDa.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Expressing, Western Blot, Infection, Transfection

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Determination of expressed full-length pGC-A functionality via whole-cell activity assay. The competitive ELISA assay measured the cGMP yield level in Sf9 cells expressing full-length pGC-A versus control Sf9 cells (n = 2). Both cell types were incubated with different concentrations of MANP ligand (0 to 10 7 pmol). Incubation with 0 pmol MANP served as a negative control.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Activity Assay, Competitive ELISA, Expressing, Incubation, Negative Control

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Purification of full-length pGC-A via affinity and size exclusion columns. ( A ) Western blot (anti-pGC-A) of samples from cell lysis to the affinity column. pGC-A (red arrow) is ~ 120 kDa. ( B ) Coomassie blue stain from cell lysis to the affinity column. ( C ) Coomassie blue stain of the eluted fraction from Superose 6 that was used for protein crystallization. M: marker; P: membrane pellet from ultracentrifugation after solubilization with n-dodecyl-β-D-maltoside (DDM) and cholesteryl hemisuccinate (CHS); FT: flowthrough from the affinity column; 50, 100, 500: imidazole (mM) at elution from the affinity column.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Purification, Western Blot, Lysis, Affinity Column, Staining, Crystallization Assay, Marker

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Presence of pGC-A in crystals was confirmed via western blot. The anti-pGC-A antibody was used in the western blot. The pGC-A monomer is 120 kDa. Lane 1: combined mix: combined crystallization drops collected in the PCR tube. Lane 2: supernatant: the supernatant collected from the centrifuged combined mix. Lane 3: washed pellet: the crystal pellet was washed with the precipitant solution and collected again via centrifugation. Lane 4: washed supernatant: the supernatant from washed crystal pellet. Lane 5: crystallization drop: one crystallization hanging drop directly mixed with SDS sample buffer. Lane 6: crystallization sample: purified pGC-A before crystallization, serving as a positive control.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Western Blot, Crystallization Assay, Centrifugation, Purification, Positive Control

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Indexed diffraction patterns from pGC-A microcrystals collected via serial crystallography at the Advanced Photon Source. Three indexable diffraction patterns with the highest resolution of 3 Å. ( A ) Blue/pink diffraction graphs show diffraction patterns analyzed using CrystFEL software. ( B ) The original diffraction patterns shown with white background. All diffraction dots were manually circled in red for better visualization.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Software

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Dynamic oligomeric states of pGC-A seen in replicate runs of Superose 6 size exclusion chromatography may be dependent on protein concentration. ( A ) The Superose 6 10/300GL column performance profile. Five standard proteins were used to generate relative molecule elution points based on different molecular sizes. Thyroglobulin (669 kDa) eluted at 14.19 mL, ferritin (440 kDa) eluted at 15.96 mL, aldolase (158 kDa) eluted at 17.58 mL, ovalbumin (44 kDa) eluted at 18.49 mL, and aprotinin (6.5 kDa) eluted at 21.60 mL. ( B-D ) Size exclusion chromatography of pGC-A. ( B ) The peak intensity at 17.3 mL corresponds to the pGC-A monomeric state (120 kDa). pGC-A monomer is the major peak determined by chromatography. Other ratios were faded out in the background and served as supplemental comparison. ( C ) pGC-A tetramer and monomer present similar ratios in the chromatographic separation. The peak intensity at 14.76 mL and 17.29 mL corresponds to pGC-A tetrameric (480 kDa) and monomeric states, respectively. ( D ) The pGC-A tetramer is the major peak. The peak intensity at 15.05 mL corresponds to the pGC-A tetrameric state.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Size-exclusion Chromatography, Protein Concentration, Chromatography

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Silver stain of high resolution clear-native PAGE from pGC-A Superose 6 fractions. Superose 6 column eluted tetramer (480 kDa) and monomer (120 kDa) size peaks were concentrated and analyzed in a 4–16% native gel. M, marker.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Silver Staining, Clear Native PAGE, Marker

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Silver stained clear-native PAGE of different oligomeric samples treated with or without dithiothreitol (DTT) overnight. Three peak samples, which represent the tetramer, dimer, and monomer of full-length pGC-A, were concentrated and split in half for treatment with or without DTT. S, concentrated sample only; S + DTT, concentrated sample incubated with 1 M DTT overnight.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Staining, Clear Native PAGE, Incubation

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Purified full-length pGC-A in vitro functional activity test. ( A ) Functional activity for two pGC-A oligomeric states with ATP incubation. The control group was GTP and ATP in sample buffer. For activity values in units of mg of purified pGC-A, the y-axis values of pmol/mL can be converted to nmol/mg protein by multiplying by 0.00873. ( B ) Functional activity for two pGC-A oligomeric states without ATP incubation. The control group was GTP only in the sample buffer. ( C ) Competitive cGMP ELISA standard fit in four parameters logistic (4PL) curve. The left Y-axis is the B/B0 (%) value and represents the percentage of bound cGMP. The right Y-axis represents the average net optical density (OD) reading at 405 nm. Both standard curves were generated with a 95% confidence interval. ( D ) The cGMP yield differences between pGC-A oligomer samples incubated with or without ATP were analyzed. All raw data points were analyzed via the ROUT method (Q = 1%) to remove significantly impossible outlier values before data analysis. One-way ANOVA was used to determine the statistical significance between samples and control in graphs ( A ) and ( B ). Two-way ANOVA was used to determine the statistical significance in graph ( D ). Each dot represented to the sample point and plotted as mean ± standard deviation (SD). * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001 and ns P ≥ 0.05, Not significant.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Purification, In Vitro, Functional Assay, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Generated, Standard Deviation

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Proposed native state and three-step mechanism of full-length pGC-A. First, the full-length pGC-A forms a tetramer complex in the native state by non-covalent interactions (e.g., hydrogen bond and hydrophobic interactions). In each tetramer complex, there are two functional units, and each functional unit may represent a dimer. The narrowest part of the tetramer is the transmembrane domain. Second, the pGC-A signal transduction mechanism is not ATP-dependent. The current ATP-dependent two-step activation mechanism should instead be three-step. The first step is ligand (ANP) binding, which moderately activates the pGC-A; the second step is binding ATP, which partially boosts protein activity; the third step is the pGC-A phosphorylation, which fully activates the guanylyl cyclase.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Functional Assay, Transduction, Activation Assay, Binding Assay, Activity Assay