hiblock buffer  (Revvity)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identification and characterization of a MAPT-targeting locked nucleic acid antisense oligonucleotide therapeutic for tauopathies

doi: 10.1016/j.omtn.2022.07.027

Figure Lengend Snippet: ASO-001933 is a highly potent and selective ASO targeting MAPT on human neurons (A) Schematic diagram of ASO treatment schedule in hESCs for (B)–(F). (B) Dose-dependent reduction of MAPT expression at RNA and protein level upon ASO-001933 or BIIB080 treatment. hESC-derived neurons were treated with ASO at indicated concentrations. RNA and protein levels were assessed by qRT-PCR and AlphaLISA respectively. Absolute IC 50 values are reported in the figure (n = 3 /treatment, mean +/- SEM). (C) Quantification of 3R and 4R Tau mRNA by qPCR after ASO treatment at 1 μM (n = 3 /treatment, mean +/- SEM). (D and E) Representative images of RNAscope ISH and ICC using probes against MAPT CDS, MAPT 3′ UTR (D) and antibody against Tau (Tau HT7) (E). (F) Volcano plot illustrating differentially regulated proteins from proteomics analysis between ASO-001933-treated cells versus NT ASO-treated neurons (n = 3). MAPT is the only significantly regulated protein (Log2 FC = −2.94, adjusted Log10 p value = −4.54). (G) Evaluation of off-target effects of ASO-001933 in hiPSC-derived neurons. Volcano plot showing differentially expressed genes (Log2FC > 1 or Log2FC< −1 and −Log10 adjusted p >20) from RNA-seq analysis after 72 h of treatment (n = 3).

Article Snippet: Cell extracts were diluted 20-fold into AlphaLISA HiBlock buffer (PerkinElmer) for assay.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, RNAscope, RNA Sequencing

Journal: Neuropsychopharmacology

Article Title: Decreased striatal adenosine A 2A -dopamine D 2 receptor heteromerization in schizophrenia

doi: 10.1038/s41386-020-00872-9

Figure Lengend Snippet: a Illustration of the specific AlphaLISA protein–protein interaction assay designed for A 2A R–D 2 R heteromer identification and quantification in native tissue; anti-guinea pig-coated acceptor beads (red sphere) were generated to capture a guinea pig anti-A 2A R antibodies bound to the receptors within the membrane extract; anti-rabbit coated donor beads (blue sphere) capture the immune complexes between the rabbit anti-D 2 R antibodies and the receptors within the membrane extract; A 2A R–D 2 R heteromerization brings donor beads into close proximity (<200 nm) to the acceptor beads. The excitation of the donor beads at 680 nm generates singlet oxygen ( 1 O 2 ) molecules triggering a chemical reaction within the acceptor beads, which results in a sharp peak of fluorescent emission at 615 nm (figure designed using image templates from Servier Medical Art https://smart.servier.com/image-set-download/ ). b The A 2A R–D 2 R interaction capacity in membranes from saline- ( n = 10) and PCP- ( n = 10) treated mouse striatum (left panel) or from postmortem control ( n = 10) and schizophrenic ( n = 10; SCZ) caudate (right panel) was determined by AlphaLISA method (see ‘Methods’); the specific AlphaLISA signal (i.e., ∆AlphaLISA) was calculated as described in the ‘Methods’ section and expressed as percentage (mean ± SEM) of either the saline-treated mice or control subjects. ** P < 0.01 and *** P < 0.001, Student’s t -test.

Article Snippet: Mouse striatal and human caudate membranes were homogenized in AlphaLISA buffer (AlphaLISA HiBlock Buffer, PerkinElmer, Waltham, MA, EEUU) and protein concentration determined.

Techniques: Protein Protein Interaction Assay, Generated, Membrane, Saline, Control

Journal: Neuropsychopharmacology

Article Title: Decreased striatal adenosine A 2A -dopamine D 2 receptor heteromerization in schizophrenia

doi: 10.1038/s41386-020-00872-9

Figure Lengend Snippet: a PPI impairment in saline- ( n = 10) and PCP-treated animals ( n = 10) with or without chronic treatment with haloperidol (Halo, 0.1 mg/kg/day for 5 days) or clozapine (Clz, 10 mg/kg/day for 5 days); results are expressed as percentage (mean ± SEM) of inhibition of ASR at the 75 dB amplitude prepulse acoustic stimulus; * P < 0.05, one-way ANOVA followed by Dunnett’s post hoc test compared to saline-treated mice. b A 2A R–D 2 R heteromerization in striatal membranes from the same animals shown in a determined by AlphaLISA method (see ‘Methods’); the specific AlphaLISA signal (i.e., ∆AlphaLISA) was calculated as described in the ‘Methods’ section and expressed as percentage (mean ± SEM) of the saline-treated mice. * P < 0.05 one-way ANOVA with Dunnett’s post hoc test when compared to saline-treated mice. c Representative immunoblot showing the expression of A 2A R, D 2 R and DAT in striatal membranes from animals from the same groups shown in a ; striatal membranes from PCP-treated mice were analyzed by SDS-PAGE (50 μg of protein/lane) and immunoblotted using guinea pig anti-A 2A R, rabbit anti-D 2 R, goat anti-DAT and rabbit anti-α-actinin antibodies (see ‘Methods’). d Relative quantification of A 2A R, D 2 R and DAT expression; the immunoblot protein bands corresponding to A 2A R, D 2 R, DAT and α-actinin from the same animals shown in a were quantified by densitometric scanning; values were normalized by the respective amount α-actinin in each lane to correct for protein loading. Results are expressed as percentage (mean ± SEM) of the corresponding saline-treated animal; *** P < 0.001, two-way ANOVA with Sidak’s post hoc test.

Article Snippet: Mouse striatal and human caudate membranes were homogenized in AlphaLISA buffer (AlphaLISA HiBlock Buffer, PerkinElmer, Waltham, MA, EEUU) and protein concentration determined.

Techniques: Saline, Inhibition, Western Blot, Expressing, SDS Page, Quantitative Proteomics