Journal: Frontiers in Molecular Neuroscience

Article Title: Cloning and Phylogenetic Analysis of NMDA Receptor Subunits NR1, NR2A and NR2B in Xenopus laevis Tadpoles

doi: 10.3389/neuro.02.004.2009

Figure Lengend Snippet: Detection of NMDAR subunits and splice variants in X. laevis tadpole brain . (A) Immunoblots for NR1, NR2A and NR2B were done on whole brain lysates of stage 47/48 tadpoles (Xen) with antibodies generated against homologous rat proteins. Immunoreactive bands of about the same size as in rat whole brain lysate (Rat) were found for all three subunits. (B) Western blots of whole cell lysates of HEK293 cells transfected with X. laevis the NR1-4a/b splice variant or NR2A cDNA, and their mock-transfected controls. (C) Cartoon of NR1 splice variants and the alternatively spliced exons (N1, C1, C2) and C-terminal ends (C2′) that they contain. (D) Cross-reactive bands for exons N1 and C1, as well as alternative C-terminal end C2′ in X. laevis were detected with antibodies to rat homologs by Western blot on whole brain extracts from stage 47/48 tadpoles and rat. No cross-reactive band was detected for C2. (E,F) C-terminal ends of NR1 mRNAs were amplified by RT-PCR and PCR with primer pairs 5′ to the spliced region and in the 3′UTR. (E) Twenty-six out of 29 nucleotide sequences contained only C2′ (Xen_NR1_C2′). They aligned perfectly to the published X. laevis NR1 sequence (Xen_NR1_X94156). (F) Two out of 29 nucleotide sequences contained C1 and C2′ (Xen_NR1_C1_C2′), indicating that NR1-3a/b also exists in X. laevis . The alignment to rat NR1-3a (Rat_NR1-3a) shows a very high degree of sequence conservation. Identical residues are marked with (*).

Article Snippet: An equal amount of protein per lane was separated by 8% SDS-PAGE and transferred onto nitrocellulose membrane that were probed with primary antibodies at 1:100–500: α-NR1, α-NR2B (both BD PharMingen), α-NR2A (crude rabbit serum JH 1817, gift from the Huganir Lab), α-NR1-C1 (crude rabbit serum JH 2079, gift from the Huganir Lab), α-NR1-N1, α-NR1-C2 and α-NR1-C2’ (all three Novus Biologicals) and incubated with the HRP-conjugated α-goat or α-mouse secondary antibody (Bio-Rad, 1:3000).

Techniques: Western Blot, Generated, Transfection, Variant Assay, Amplification, Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Human mutation

Article Title: Aberrant RNA splicing is the major pathogenic effect in a knock-in mouse model of the dominantly inherited c.1430A>G human RPE65 mutation

doi: 10.1002/humu.23706

Figure Lengend Snippet: Generation of c.1430A>G KI mouse. (A) Mouse genomic context for NM_029987.2:c.1430A>G mutation. DNA sequences around the mutation sites are specified along with the translated amino acid sequence in WT Rpe65. The nucleotides altered in the KI mouse model are in italics and the pathogenic A-to-G mutation is marked in red. Cas9 mRNA, gRNA targeting the point mutation site (blue arrow), and a 169-nt oligo (red arrow) carrying the nucleotide mutations were microinjected into mouse zygotes. (B) The knockin allele was identified by sequencing the PCR amplicon surrounding the mutation site (primer locations shown as green arrows in A). (C) Schemes for using ddPCR for genotyping the KI mice. A FAM-labeled probe (orange bar with T) centered on the mutation sites was designed to specifically recognize the mutant allele, while a HEX-labeled probe (green bar with R) located outside of the mutation sites served as a reference probe. The WT allele results in HEX-positive-only droplets (left panel) while the mutant allele produce double-positive droplets in both heterozygous (middle panel) and homozygous (right panel) KI mice.

Article Snippet: Genomic DNA isolated from mouse tails were analyzed using a QX200 Droplet Digital PCR (ddPCR) System (Bio‐Rad, Hercules, CA, USA).

Techniques: Mutagenesis, Sequencing, Knock-In, Amplification, Labeling

Journal: Human mutation

Article Title: Aberrant RNA splicing is the major pathogenic effect in a knock-in mouse model of the dominantly inherited c.1430A>G human RPE65 mutation

doi: 10.1002/humu.23706

Figure Lengend Snippet: The c.1430A>G mutation generates an alternative splicing site in the mouse KI allele. (A) Scheme of Rpe65 mRNA and 3’-RACE. The asterisk marks the c.1430A>G mutation. Pink bars denote positions of the Taqman assays for mRNA level quantification. Following reverse transcription, 3’-RACE was performed using forward primer (green arrow) located in Exon 6. (B) ddPCR analysis showed that Rpe65 mRNA levels are significantly lower in the KI/KI mice than in the WT. However, the expression level difference between the WT and KI/KI varies with the locations of the Taqman probes. Asterisks denote the differences are statistically significant with p<0.05 analyzed by student’s t-test; n=3. (C) Electrophoresis of the 3’-RACE products. In the WT animals, 3’-RACE yields a major band of 1.2 kb. In contrast, the 3’-RACE for the KI/KI mice result in multiple bands of lower intensity. Arrows indicate the PCR amplicons from 3’-RACE that were subsequently gel-purified and sequenced. (D) Examples of alternatively spliced transcripts cloned from the 3’-RACE of the KI/KI mice. (E) The novel transcripts identified by RACE were confirmed by ddPCR using Taqman probes specifically targeting different variants (shown as pink bars in D).

Article Snippet: Genomic DNA isolated from mouse tails were analyzed using a QX200 Droplet Digital PCR (ddPCR) System (Bio‐Rad, Hercules, CA, USA).

Techniques: Mutagenesis, Expressing, Electrophoresis, Purification, Clone Assay

Journal: Human mutation

Article Title: Aberrant RNA splicing is the major pathogenic effect in a knock-in mouse model of the dominantly inherited c.1430A>G human RPE65 mutation

doi: 10.1002/humu.23706

Figure Lengend Snippet: Defects in splicing are readily detected in human RPE65 minigene carrying the c.1430G mutation. (A) Scores of splicing signals strength for Exons 9–14 of WT RPE65 in mouse and human. High score indicates a strong splicing signal with the default cut-off score for an effective splicing site being 0.4. Note that in both mouse and human, Exon 13 has a weak splicing strength as the scores are below 0.4 and are shown in italic bold fonts. (B-E) minigene constructs that include the human genomic sequence of either WT (c.1430A) or mutants (c.1430G, c.1430C, c.1430T) were transfected into HEK293T cells and the transcribed variants were analyzed via ddPCR. One dimensional ddPCR plots for quantification of the regular spliced read-through transcript E13-E14 (B) , and the alternatively spliced variant E13-AS (C) that lacks the first 91 nucleotide of Exon 13. Data were derived from duplicates of construct transfections. Each point represents a single droplet, which is scored as positive (blue colored) or negative (grey) depending on the fluorescent amplitude. The expression levels were normalized to the expression of reference gene HPRT1 (D and E). While the read-through transcript E13-E14 was readily detected in both the cells expressing WT and the mutant minigenes (B and D), the alternative spliced E13-AS was only enriched in the cells expressing mutant minigene (C and E). The asterisk marks the c.1430A>G mutation. Pink bars denote positions of the Taqman probes for mRNA level quantification.

Article Snippet: Genomic DNA isolated from mouse tails were analyzed using a QX200 Droplet Digital PCR (ddPCR) System (Bio‐Rad, Hercules, CA, USA).

Techniques: Mutagenesis, Construct, Sequencing, Transfection, Variant Assay, Derivative Assay, Expressing

Journal: eLife

Article Title: Induction of osteogenesis by bone-targeted Notch activation

doi: 10.7554/eLife.60183

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , iScript cDNA Synthesis Kit , BIO-RAD , Cat# 170–8891 , .

Techniques: Recombinant, Variant Assay, Plasmid Preparation, Sequencing, Control, Gene Expression, Bicinchoninic Acid Protein Assay, cDNA Synthesis, Amplification, Magnetic Beads, Protease Inhibitor, Western Blot, Software, Staining

Journal: Journal of Diabetes and Metabolic Disorders

Article Title: A case-control study to determination FBXW7 and Fetuin-A levels in patients with type 2 diabetes in Iraq

doi: 10.1007/s40200-021-00738-x

Figure Lengend Snippet: Clinical data in patients having diabetes mellitus (GI newly diagnosed DM, GII DM with metformin therapy) and control group

Article Snippet: While HbA1c was determined using Bio- Rad VARIANT Hemoglobin A1c programmer.

Techniques: Control, Significance Assay

Journal: Journal of Diabetes and Metabolic Disorders

Article Title: A case-control study to determination FBXW7 and Fetuin-A levels in patients with type 2 diabetes in Iraq

doi: 10.1007/s40200-021-00738-x

Figure Lengend Snippet: Correlation analysis of variables associated with serum FBXW7 protein level in the study populations

Article Snippet: While HbA1c was determined using Bio- Rad VARIANT Hemoglobin A1c programmer.

Techniques: Control

Journal:

Article Title: Amino Acid Substitutions at Ambler Position Gly238 in the SHV-1 ?-Lactamase: Exploring Sequence Requirements for Resistance to Penicillins and Cephalosporins

doi: 10.1128/AAC.46.12.3971-3977.2002

Figure Lengend Snippet: Western blot and slot blot of SHV-1 β-lactamase and mutants at the 238 position. The Western blot and slot blot were probed with 1 μg of anti-SHV-1 antibody/ml and horseradish peroxidase-conjugated protein G (7). Single-letter designations for the Gly238 mutant β-lactamases are listed above each slot. SK− represents the strain E. coli DH10B with the vector pBCSK(−) without the SHV β-lactamase. (Inset) Western blot of SHV-1 and Gly238Ala variant of the SHV β-lactamase. Equal amounts of purified β-lactamase were loaded in each lane. Lane 1, SHV-1; lane 2, Gly238Ala. As evaluated by densitometry, SHV-1 and Gly238Ala are nearly equivalent.

Article Snippet: Each of the strains possessing the 19 variants and the wild-type β-lactamase were assayed for in vivo steady-state expression levels by probing them with 1 μg/ml of purified anti-SHV antibody and horseradish peroxidase-conjugated protein G (Bio-Rad) as previously reported ( 7 ).

Techniques: Western Blot, Dot Blot, Mutagenesis, Plasmid Preparation, Variant Assay, Purification