Journal: Nature Communications

Article Title: Small molecule splicing modifiers with systemic HTT-lowering activity

doi: 10.1038/s41467-021-27157-z

Figure Lengend Snippet: a 5′ splice sites (ss) sequence at regions (−4 to −1 and +1 to +6) were studied for the enrichment of Inc (included) vs. NC (no change) exons. Significance scores are shown. Wide boxes represent exons. b Schematic of 5′ss sequence logo in the three exon groups: annotated NC, annotated (inclusion) Inc and psiExons Inc. c Diagram illustrating the design of huntingtin (HTT) minigene constructs for studying compound-induced splicing. d Polymerase chain reaction (PCR) analysis of RNA extracts from HEK293 cells transfected with wild-type (wt) human HTT minigene or constructs with point mutations in the −2 to +3 region of the 5′ss; cells were treated with dimethyl sulphoxide (DMSO) or HTT-C2 (0.010–1 µM). The data are from a single transfection experiment with multiple concentrations tested for a given construct; the “WT” control construct has been used multiple times ( n > 3). e Sequence of the 20-nucleotide region upstream of the 5′ss of HTT stop-codon psiExon49a showing partial deletions and mutations performed on this region, and their effects on HTT-C2 induced splicing (lower panel). Through partial deletion or mutation of the nucleotides CAGGA at positions −38 to −34, this region was shown to be important in regulating splicing events. The data are from a single transfection experiment with multiple concentrations tested for a given construct; the “WT” control construct has been used multiple times ( n > 3).

Article Snippet: Human B-lymphocytes and fibroblasts derived from the same homozygous patient with Huntington’s disease (HD) (GM04856/GM04857) and a healthy donor (GM07492/GM07491) (Coriell Institute for Medical Research), human neuroblastoma (SH-SY5Y) cells (ATCC ® ), human embryonic kidney 293 (HEK293) cells (ATCC); Madin–Darby Canine Kidney (MDCK) cells (ATCC ® ); MDCK cells expressing multidrug-resistance mutation 1 (MDCK-MDR1) (Absorption Systems); mouse CT26 cells (ATCC) were all grown at 37 °C in a humidified 5% CO 2 atmosphere.

Techniques: Sequencing, Construct, Polymerase Chain Reaction, Transfection, Control, Mutagenesis

Journal: Journal of Neuroscience

Article Title: Glutamate Receptor Trafficking: Endoplasmic Reticulum Quality Control Involves Ligand Binding and Receptor Function

doi: 10.1523/jneurosci.4573-04.2005

Figure Lengend Snippet: Figure 2. [ 3H]Kainate binding and functional assessment by patch-clamp recording of GluR6-binding site mutants. A, Saturation analysis of [ 3H]kainate binding to GluR6-wt ex- pressedinHEK293cellsyieldedaKDof36nMandaBmaxof3.8pmol/mgprotein.Specificbinding wasdeterminedinthepresence(nonspecific)andabsence(total)of1mMglutamate.B,Specific binding of 100 nM [ 3H]kainate to GluR6-wt, R523G, T690G, E738G, and E738D (60 g of pro- tein). Western blots confirmed similar amounts of C-GluR6 immunoreactivity in 25 g of pro- tein used for the binding assays (inset). C, Outside-out patches were pulled from HEK293 cells expressing GluR6-wt, R523G, T690G, or E738G, voltage clamped at 70 mV, and tested by ultrafast application of 3 or 30 mM glutamate (GLU). For GluR6-wt, 3 mM glutamate was satu- rating and elicited large peak currents (2.8 0.4 nA). No currents were detected in patches from cells expressing R523G, T690G, or E738G. Application of 3 or 30 mM glutamate demon- stratesthatE738Dreceptorsarefunctional,whereas3mMglutamateisnolongersaturatingfor this mutant. D, Dose–response curve showing the rightward shift of the EC50 value ( 100- fold) for the mutation E738D compared with GluR6-wt in peak response to glutamate.

Article Snippet: Human embryonic kidney 293 (HEK293) fibroblasts (CRL 1573; American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium supplemented with 10% fetal bovine serum and 2 mM glutamine (Invitrogen, Gaithersburg, MD) and maintained at 37°C in a 5% CO2 environment.

Techniques: Binding Assay, Functional Assay, Patch Clamp, Western Blot, Expressing, Mutagenesis

Journal: Journal of Neuroscience

Article Title: Glutamate Receptor Trafficking: Endoplasmic Reticulum Quality Control Involves Ligand Binding and Receptor Function

doi: 10.1523/jneurosci.4573-04.2005

Figure Lengend Snippet: Figure 3. Cell-surface expression of the GluR6-binding site mutants. A, Confocal images of extracellular N-GluR6 (green) and intracellular C-GluR6 (red) immunofluorescence staining in HEK293 cells expressing GluR6-wt and GluR6-binding site mutants. N-GluR6 staining before cell membrane permeabilization demonstrates robust surface expression of GluR6-wt and E738D. In contrast, the absence of N-GluR6 staining indicates that R523G, T690G, and E738G are not present at the cell surface. C-GluR6 staining was evident for GluR6-wt and GluR6 mutants after permeabilization. Staining of nontransfected controls was negative. B,PrimaryculturesofratastrocytestransfectedwithGluR6-wtorT690GwerestainedforN-andC-GluR6asabove.C,Biotinylated cell-surfaceproteins(50g)wereprecipitatedwithNeutrAvidinbeads.Onlytraceamounts,ifany,ofR523G,T690G,andE738G werefoundatthecellsurface.GluR6-wtandE738Dwerebothisolatedinthebiotinylatedfraction.Thepercentageofcell-surface expressionofimmunoreactiveGluR6wasdeterminedbydensitometryusing10goftotalprotein.Theblotisrepresentativeof three separate experiments. IB, Immunoblot; IP, immunoprecipitate. D, Biotinylation studies, using GluR1-wt as a control, re- vealed that a homologous GluR1-binding site mutant, R499G, also fails to reach the cell surface.

Article Snippet: Human embryonic kidney 293 (HEK293) fibroblasts (CRL 1573; American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium supplemented with 10% fetal bovine serum and 2 mM glutamine (Invitrogen, Gaithersburg, MD) and maintained at 37°C in a 5% CO2 environment.

Techniques: Expressing, Binding Assay, Immunofluorescence, Staining, Membrane, Western Blot, Control, Mutagenesis

Journal: Journal of Neuroscience

Article Title: Glutamate Receptor Trafficking: Endoplasmic Reticulum Quality Control Involves Ligand Binding and Receptor Function

doi: 10.1523/jneurosci.4573-04.2005

Figure Lengend Snippet: Figure4. ERretentionandglycosylationstateofGluR6-bindingsitemutants.A,ConfocalimagesofintracellularC-GluR6(red) immunofluorescence staining in HEK293 cells coexpressing the EYFP-ER marker and GluR6-wt or GluR6-binding site mutants. GluR6-wt and E738D demonstrate robust surface staining that is not colocalized with EYFP-ER. In contrast, R523G, T690G, and E738G are not present at the cell surface and are colocalized entirely with EYFP-ER. The merged images show the overlapping localization of C-GluR6 and EYFP-ER markers. Identical results were obtained with antibodies to other ER markers, calnexin and calreticulin. B, Representative confocal images of the GluR6-wt (top) or the GluR6-binding-deficient mutant R523G (bottom) cotransfectedwiththeEYFP-Golgimarker.Themergedimageofimmunofluorescencestainingdemonstratesthelackofcolocal- ization of R523G (red) and the EYFP-Golgi marker (green). C, Triton X-100 (0.5%) solubilized lysates of HEK293 cells transfected withGluR6-wtorGluR6-bindingsitemutantsweresubjectedtoEndoHdigest.Westernblotanalysisofcontrol(Gly)andEndoH- digested(Cut)samples(15–30gofprotein)revealsthatR523G,T690G,andE738GareentirelyEndoHsensitive.Incontrast,the majorityofGluR6-wtandE738DareEndoHinsensitive.D,ParalleldigestsdemonstratePNGaseFandEndoHdeglycoslateR523G, T690G,andE738Gtothesameextent,confirmingtheirimmatureN-linkedglycosylationstate.Theblotsarerepresentativeofat least three separate experiments.

Article Snippet: Human embryonic kidney 293 (HEK293) fibroblasts (CRL 1573; American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium supplemented with 10% fetal bovine serum and 2 mM glutamine (Invitrogen, Gaithersburg, MD) and maintained at 37°C in a 5% CO2 environment.

Techniques: Immunofluorescence, Staining, Marker, Binding Assay, Mutagenesis, Transfection