mouse anti sptbn2  (Santa Cruz Biotechnology)

Journal: Cell reports methods

Article Title: Highly efficient manipulation of nervous system gene expression with NEPTUNE

doi: 10.1016/j.crmeth.2021.100043

Figure Lengend Snippet: NEPTUNE with virus encoding shSptbn2-H2B-GFP or Scr-H2B-GFP , and embryos collected at E9.5, E13.5, or E18.5 (A) qPCR for Sptbn2 in GFP + cells sorted from whole embryos at E9.5. Two Scr-H2B-GFP embryos were pooled per “Scr” sample (each dot = 2 embryos). Three to four shSptbn2-H2B-GFP embryos were pooled per sample (each dot = 3 or 4 embryos). (B) At E9.5, shSptbn2-H2B-GFP embryos presented with three phenotypic severities; 17% are resorbed. Class 1 phenotype (22%) was defined as a straightened body axis, Class 2 phenotype (28%) as a body with a rightward-skewed lumbar and sacral region, and Class 3 phenotype (33%) as developmentally delayed and failure to undergo embryonic turning. (C) Dorsal views of neural tube of control Scr-H2B-GFP - and shSptbn2-H2B-GFP- injected embryos from the three classes. In control, arrowheads denote a straight spinal cord with even closure. In Class 1–3 shSptbn2-H2B-GFP embryos, arrowheads denote kinks in the spinal cord with uneven closure. (D and E) Western blot and quantification of SPTBN2 in whole embryos injected with Scr-H2B-GFP or shSptbn2-H2B-GFP . Embryos were pooled for analysis, two embryos per Scr-H2B-GFP lane and three per shSptbn2-H2B-GFP lane. Equal protein amounts were loaded. (D) Western blot for SPTBN2. (E) Quantification of SPTBN2 levels in (D), normalized to ACTB. n (number of embryos included in analysis, contributing to pools) is depicted below each condition. SPTBN2 levels are inversely correlated with phenotype severity. (F) In situ hybridization for FoxA2 in Scr-H2B-GFP or shSptbn2-H2B-GFP E9.5 embryos. White arrowheads denote an angulated flexure point in Class 1 and Class 2 embryos. Yellow arrowhead denotes the failed turning point in a Class 3 embryo. Red arrowhead denotes an interruption in floorplate Foxa2 in the Class 3 embryo. Brackets denote straightened portions of neural tube. (G) Survival of injected embryos per injected litter in Scr-H2B-GFP - or shSptbn2-H2B-GFP -injected litters at E9.5, E13.5, and E18.5. Each dot represents the percentage survival in one litter. The total number of injected embryos/amniotic cavities is depicted under the graph. No shSptbn2-H2B-GFP embryos survived until E18.5. (H) Dorsal and semi-ventral views of an E13.5 shSptbn2-H2B-GFP embryo demonstrates rightward skewing and an abdominal wall closure defect with externalized internal organs including the liver. (I) Sections of embryos in (F) from thoracic to sacral spinal cord. Boxed regions depict a wider floorplate in Class 2 embryos (red arrowheads), and a wider floorplate consisting of a single layer of cells in the lumbar region of Class 3 embryos (red arrowheads in lumbar boxed region). Note the skewed spinal cord floorplate compared with the body axis in Class 3. H, heart; G, gut. (J) Expression of cleaved CASPASE 3 (cCASP3) in E9.5 spinal cord (representative of n = 3 for Scr-H2B-GFP ; n = 6 for shSptbn2-H2B-GFP Class 2 and 3, respectively). Scale bars represent 50 μm. Differences in expression levels in (A) were analyzed with one-way ANOVA and Dunnett’s multiple comparisons test, ****p < 0.0001. Differences in (D) were not tested statistically because two pools per condition should not be statistically tested. Differences in (G) were not tested statistically because only one litter was included at E13.5, due to an obvious impact on survival, and 3R considerations precluded further experiments.

Article Snippet: Mouse monoclonal anti-Sptbn2 , Abcam , Ab238055.

Techniques: Injection, Western Blot, In Situ Hybridization, Expressing

Journal: Cell reports methods

Article Title: Highly efficient manipulation of nervous system gene expression with NEPTUNE

doi: 10.1016/j.crmeth.2021.100043

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Sptbn2 , Abcam , Ab238055.

Techniques: Recombinant, Electron Microscopy, Titration, Ligation, Gel Extraction, Labeling, Plasmid Preparation, Purification, Western Blot, Stripping, shRNA, Clone Assay, Software, Imaging

Journal: Nature Communications

Article Title: EMX2-GPR156-Gαi reverses hair cell orientation in mechanosensory epithelia

doi: 10.1038/s41467-021-22997-1

Figure Lengend Snippet: a Schemes representing a single auditory HC from the lateral/abneural (top) or medial/neural (bottom) side, and HC orientation (arrows) in the auditory epithelium and the utricular and saccular maculae. In the maculae, two HC populations of opposing orientations are separated by a virtual line of polarity reversal (LPR, yellow dashed line). Domains where HC orientation was quantified in e , f are indicated in blue (utricle: lateral extrastriolar (LES), LPR and medial (M) domains; saccule: anterior (ANT), LPR and posterior (POST) domains). b Phylogenetic tree of class C GPCRs adapted from . c , d LPR region in P2 utricle ( c ) and saccule ( d ). Top panels show a low magnification view with SPTBN2 (βII-spectrin) labeling revealing HC orientation by the position of the off-center fonticulus devoid of signal. Bottom panels show a distinct region at higher magnification where PCNT (Pericentrin) labels the basal body below the fonticulus. The LPR can be traced in controls but not in mutants, where all HCs generally point laterally in the utricle and anteriorly in the saccule. e , f Circular histograms of HC orientation by region in the utricle ( e ) and saccule ( f ). Histograms show frequency distribution at P0-P2 (10° bins in a referential where 90° (top) is lateral in the utricle and anterior in the saccule; n indicates HC number in N = 4 animals; Watson U2 test of homogeneity; normalized difference (ND) value indicates how many standard deviations separate the circular means of each distribution). PTXa indicates the Cre-inducible R26-LSL-PTXa allele. Littermate controls for FoxG1-Cre ; PTXa are Cre-negative PTXa animals. Arrows indicate HC orientation. Scale bars are 20 µm ( c , d top), 5 µm ( c , d bottom).

Article Snippet: Primary antibodies used were: goat anti-GPR156 (Santa Cruz; sc102572; TCA; 1:100), rabbit anti-GPR156 (Novus; NBP1-83402; TCA; 1:100), mouse anti-acetylated Tubulin (Santa Cruz; 23950; PFA; 1:500), rabbit anti-pericentrin/PCNT (Biolegend; PRB-432C; PFA; 1:400), mouse anti-βII-Spectrin/SPTBN2 (BD Transduction Lab; 612562, PFA; 1:200), rat anti-ZO1 (Developmental Studies Hybridoma Bank; R26.4C; TCA; 1:200), rabbit anti-Gαi3 (Santa Cruz; sc-262; PFA; 1:400), chicken anti-Gαi3 (Sigma; GW22489; PFA; 1:400; used for cochlear explants), rabbit anti-DAPLE/CCDC88C (Proteintech; 25769-1-AP; TCA; 1:400), mouse anti-MYO7A (Developmental Studies Hybridoma Bank; 138-1; PFA; 1:500), goat anti-FZD6 (R&D Systems; AF1526; PFA; 1:200), rabbit anti-VANGL2 (gift from Philippe Gros, McGill University; PFA; 1:500), goat anti-GPSM2/LGN (ThermoFisher Scientific; PA5-18646; PFA; 1:200), rabbit anti-βGalactosidase (Cappel discontinued aliquot; PFA; 1:1000; now MP Biomedical 55976), mouse anti-aPKC/PRKCZ (Santa Cruz; sc-216; PFA; 1:100), rabbit anti-PARD6B (Santa Cruz; sc-67393; PFA; 1:100), rabbit anti-PARD3A (Proteintech; 11085-1-AP; PFA; 1:200), rabbit anti-CALB1 (Cedarlane/Millipore; AB1778(CH); PFA; 1:500), goat anti-SPP1/Osteopontin (R&D Systems; AF808; PFA; 1:100), goat anti-SOX2 (Santa Cruz; 17320; PFA; 1:500), rabbit anti-EMX2 (Trans Genic; KO609; PFA; 1:250).

Techniques: Labeling