Journal: International Journal of Molecular Sciences

Article Title: In Situ synNotch-Programmed Astrocytes Sense and Attenuate Neuronal Apoptosis

doi: 10.3390/ijms26094343

Figure Lengend Snippet: Schematic representation of the dual-synNotch system for sensing neuronal apoptosis. Astrocytes are genetically programmed to express two synNotch receptors on their surface, which specifically recognize the apoptosis marker PS and the neuronal marker ganglioside Gt1b. Upon activation, the synNotch receptor that binds to PS releases TetR-VP64 (tTA) into the nucleus, thereby initiating transcription of Gal4-DBD and EGFP. When activated by binding with Gt1b, the second synNotch receptor releases VP64-AD into the nucleus. VP64-AD then interacts with Gal4-DBD through complementary leucine zipper sequences to form a complete transcription factor, which subsequently initiates the transcription and expression of GLuc-IgG1 Fc and BDNF-Flag.

Article Snippet: The response element downstream of the synNotch receptor contained an inducible promoter covering five copies of the Gal4 DBD targeting the UAS (5′-GGAGCACTGTCCTCCGAACG-3′) or seven copies of the TetR targeting the TRE sequence (5′-TCCCTATCAGTGATAGAGA-3′), followed by a minimal CMV promoter (Addgene plasmid, #79126).

Techniques: Marker, Activation Assay, Binding Assay, Expressing

Journal: Nature methods

Article Title: Chemogenetic control of gene expression and cell signaling with antiviral drugs

doi: 10.1038/s41592-018-0042-y

Figure Lengend Snippet: (a) Design of the drug-inducible “turn-on” TFs. (b) Western blot showing accumulation of full-length Gal4DB-NS3-Gal4TA (anti-HA; 60.6 kDa) in response to BILN-2061, and corresponding H2B-Citrine expression (anti-H2B; 46.5 kDa). Endogenous histone H2B (13.8 kDa) served as a loading control. (c) Fluorescence images of CHO-derived reporter cells (UAS H2B-Citrine) stably expressing Gal4DB-NS3-Gal4TA in the absence and presence of 3 μM BILN-2061. Scale bar is 50 μm. (d) H2B-Citrine fluorescence as determined by flow cytometry in HEK 293FT cells transiently expressing TFs with the indicated TA domains and treated with varying concentrations of BILN-2061. (e) General design of the “turn-off” TFs containing a generic membrane-localizing element (green). (f) Fluorescence images of HEK 293A cells transiently expressing BFP-TMD-NS3-Gal4-mCherry. BFP is N-terminal to the TMD; Gal4 is tagged with mCherry. Scale bar is 10 μm. (g) Immunostained HeLa cells showing drug-induced membrane-targeting of Gal4min via myr-palm-NS3-Gal4min. Drug is 3 μM BILN-2061. Scale bar is 25 μm. (h) Fluorescence emission from HEK 293FT containing UAS H2B-Citrine and TRE BFP reporter constructs and transiently coexpressing rTetR-NS3-VP64-p65 and TMD-NS3-Gal4min. Cells were treated with 100 ng/mL doxycycline and varying concentrations of BILN-2061. Negative controls correspond to background activation of each reporter in cells containing mismatched TFs. Values plotted in (d) and (h) represent means +/− s.d., n = 3 biologically independent samples based on geometric mean fluorescence determined by flow cytometry.”

Article Snippet: DNA constructs Plasmid DNA and detailed sequence information for expression vectors encoding Gal4 DB -NS3-Gal4 TA , Gal4 DB -NS3-VP64, Gal4 DB -NS3-VP64-p65, rTetR-NS3-VP64-p65, myr-palm-NS3-Gal4 min , dCas9-NS3-NLS/VPR, and SNAP-dCas9-NS3-NLS/VPR can be obtained via AddGene.

Techniques: Western Blot, Expressing, Control, Fluorescence, Derivative Assay, Stable Transfection, Flow Cytometry, Membrane, Construct, Activation Assay

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

Journal: IBRO Neuroscience Reports

Article Title: Non-canonical C-terminal variant of MeCP2 R344W exhibits enhanced degradation rate

doi: 10.1016/j.ibneur.2023.09.007

Figure Lengend Snippet: Characterization of an uncanonical MECP2 variant. (A). The patient at the age of 3. (B). The patient at the age of 18. Mild dysmorphology, including cupids bow, mild upslanting palpebral fissures, tubular nose, progressed over time. (C). Forward and reverse sequences of MECP2 exon 4 of the patient. One allele of MECP2 contains a reported disease-causing variant c.1030C>T in the patient. Both sequences are aligned to the reference genomic sequence of MECP2 from NCBI GenBank. (D). Construction of MECP2-WT and MECP2-R344W variant plasmids.

Article Snippet: The coding-region of wild type (WT) MECP2 gene was obtained from the pCMX-Gal4-hMECP2 plasmid (Addgene, #48082), a gift from Dr. Huda Zoghbi.

Techniques: Variant Assay, Sequencing

Journal: IBRO Neuroscience Reports

Article Title: Non-canonical C-terminal variant of MeCP2 R344W exhibits enhanced degradation rate

doi: 10.1016/j.ibneur.2023.09.007

Figure Lengend Snippet: MECP2-R344W mutation decrease the stability of MECP2 protein. (A) and (B). Decreased MECP2-R344W protein levels following 9 h of CHX treatment. (A.) Western blot analysis of MECP2 protein levels in MECP2 transfected HEK293T cells following 0, 3, 6, 9 h CHX treatment. (B). Grey bars: MECP2-WT protein levels. Red bars: MECP2-R344W protein levels. Values are normalized to α-tubulin of each timepoint. Two-way ANOVA was performed. P = 0.0198. All error bars represent the mean ± SEM (n = 5).

Article Snippet: The coding-region of wild type (WT) MECP2 gene was obtained from the pCMX-Gal4-hMECP2 plasmid (Addgene, #48082), a gift from Dr. Huda Zoghbi.

Techniques: Mutagenesis, Western Blot, Transfection

Journal: IBRO Neuroscience Reports

Article Title: Non-canonical C-terminal variant of MeCP2 R344W exhibits enhanced degradation rate

doi: 10.1016/j.ibneur.2023.09.007

Figure Lengend Snippet: Blocking proteosome pathway can restore MECP2-R344W protein level. (A) and (B). MG132 treatment rescued FLAG-tagged MECP2-R344W protein degradation. (A). Western blot analysis of MECP2 protein levels of FLAG-tagged MECP2-WT and MECP2-R344W following 9 h CHX treatment with or without MG132. (B). Values are represented as the fold change relative to cells treated with 9 h CHX treatment but without MG132. MECP2-R344W protein level was restored 50% by MG132 compared to untreated group while no rescue effect was observed in MECP2-WT protein levels with 9 h treatment of MG132. Paired t-test was performed. P = 0.0411. All error bars represent the mean ± SEM (n = 4).

Article Snippet: The coding-region of wild type (WT) MECP2 gene was obtained from the pCMX-Gal4-hMECP2 plasmid (Addgene, #48082), a gift from Dr. Huda Zoghbi.

Techniques: Blocking Assay, Western Blot

Journal: IBRO Neuroscience Reports

Article Title: Non-canonical C-terminal variant of MeCP2 R344W exhibits enhanced degradation rate

doi: 10.1016/j.ibneur.2023.09.007

Figure Lengend Snippet: NCoR/SMRT interaction and nuclear localization isn’t interfered by MECP2-R344W mutation. (A) and (B). Undisrupted MECP2-R344W protein interaction with NCoR/SMRT complex components. (A). Co-immunoprecipitation of TBLR1 and HDAC3 from extracts of MECP2-WT and MECP2-R344W transfected HEK293T cells. (B). Values are presented as “Input” divided by “IP” and are shown as the fold change relative to MECP2-WT. Unpaired t-test was performed. All error bars represent the mean ± SEM (n = 3). (C) and (D). Similar MECP2 nuclear localization pattern between MECP2-WT and MECP2-R344W proteins. Mouse neuroblastoma N2a cells that transiently expressed the full-length EGFP-tagged MECP2-WT and MECP2-R344W, representing the co-localization at chromocenters (DAPI; Blue) and the recombinant EGFP-tagged MECP2 protein (Green). Scale bar: 5 µm. (D) . Average mean of the Pearson Correlation Coefficient (PCC) values (n = 20 cells for WT and n = 29 for R344W; ± SEM shown). Unpaired t-test was performed.

Article Snippet: The coding-region of wild type (WT) MECP2 gene was obtained from the pCMX-Gal4-hMECP2 plasmid (Addgene, #48082), a gift from Dr. Huda Zoghbi.

Techniques: Mutagenesis, Immunoprecipitation, Transfection, Recombinant