human mouse sonic hedgehog shh n terminus antibody  (R&D Systems)

Journal: eLife

Article Title: The ciliary kinesin KIF7 controls the development of the cerebral cortex by acting differentially on SHH signaling in dorsal and ventral forebrain

doi: 10.7554/eLife.100328

Figure Lengend Snippet: ( A–C ) and distribution of Sonic Hedgehog (SHH) protein ( D ) in the embryonic stage 13.5 (E13.5)-E14.5 forebrain. ( A, B ) Distribution in median ( A ) and caudal ( B ) coronal sections of Shh mRNA detected by in situ hybridization with an antisense Shh probe at E13.5 ( A1, B1 ) and by RNAscope at E14.5 (A2-3, B2). Shh transcripts are strongly expressed in the medial ventral forebrain (SVZ and mantle zone of the MGE and septum, A1, A2 ), in the mantle zone of the CGE ( B1, B2 ), in the zona limitans intrathalamica (ZLI in B1) and in the ventral midline of the third ventricle (V3 in B1). RNAscope further confirmed the strong expression of Shh mRNA (A3, green) in MGE and septum regions that strongly express Lhx-6 mRNA (A2, red). Confocal observations in the SVZ and mantle zone of the MGE showed that Shh mRNA (green) is co-expressed with the Lhx6 mRNA (red) in a significant number of cIN (yellow cells in A4). ( C ) Confocal analyses at higher magnification of the double detection by RNAscope of Shh and Lhx-6 mRNA. Cells were identified on stacked images (△z=1 µm) using Nomarski optic. In the lateral cortex close to the PSB (C1, z-projection of 10 confocal planes) and in the dorsal cortex (C2, z-projection of 10 confocal planes), a very small proportion of cells expressing Lhx-6 mRNA also express Shh mRNA (white arrows in C1,C2). Counting in the deep stream (SVZ-IZ) and in the MZ is shown in graph C3 (9–17 fields in three sections). A few progenitors in the cortical VZ express Shh mRNA at very low levels (arrowhead, C1 ). ( D ) SHH-Nter and TBR2 co-immunostaining of Nkx2.1-Cre/R26R-tdTomato brain coronal sections at E14.5. Representative confocal merged stacked images (△z=0.2 µm; 48 images) in the pallium-subpallium boundary (PSB, D1 ), lateral cortex ( D2 ) and dorsal ( D3 ) cortex revealing SHH-Nter immunostaining in blood vessels and the presence of numerous bright dots all over the cortical neuropile. On the ventricular side of the PSB and of the lateral-most part of the LGE, SHH-Nter(+) bright elements are aligned radially. In the lateral cortical neuropile, smaller bright dots align radially in the VZ, tangentially in the SVZ-IZ, and radially in the CP (see higher magnification on the right panel in which SHH-Nter immunostaining is shown in white and the contrast is increased). Cx, cortex; LGE, MGE, and CGE, lateral, medial, and caudal ganglionic eminence; CP, cortical plate; Hyp, hypothalamus; PSB, pallium-subpallium boundary; Sp, septum; Th, thalamus; V3, third ventricle; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; MZ, marginal zone. Scale bars: 500 µm ( A, B ), 20 µm ( C ), 100 µm ( D ).

Article Snippet: Primary antibodies were goat anti SHH-Nter (1:100, R&D Systems AF464, RRID: AB_355373 ), goat anti Netrin G1a (NG1a) (1:100, R&D Systems AF1166, RRID: AB_2154822 ), rabbit anti TBR1 (1:1000, Abcam ab31940, RRID: AB_2200219 ), rabbit anti TBR2 (1:1000, Abcam ab23345, RRID: AB_778267 ), rabbit anti PAX6 (1:100, clone poly19013, Covance PRB-278P, RRID: AB_291612 ), rabbit anti GSH2 (1:2000, Millipore ABN162, RRID: AB_11214376 ), chicken anti MAP2 (1:500, Novus, NB30213), mouse anti SMI-32 (1:500, Covance SMI-32R, RRID: AB_509997 ), and rat CTIP2 (1:1000, Abcam ab18465, RRID: AB_2064130 ).

Techniques: In Situ Hybridization, RNAscope, Expressing, Immunostaining

Journal: eLife

Article Title: The ciliary kinesin KIF7 controls the development of the cerebral cortex by acting differentially on SHH signaling in dorsal and ventral forebrain

doi: 10.7554/eLife.100328

Figure Lengend Snippet: ( A, B ) Representative pictures of SHH-Nter immunostaining of C57Bl/6 mice brain coronal sections at embryonic stage 12.5 (E12.5) ( A ) and E14.5 ( B ) imaged with a macroscope. High signal is observed along the third ventricle (A1, arrow) and in the ZLI (A2, arrow) at E12.5 and in the choroid plexus and the septum at E14.5 ( B ), whereas a faint signal is observed in the cortex. ( C ) Representative picture of SHH-Nter immunostaining in the cortex at E12.5. Z stacks of 48 images confocal images (△z=0.2 µm) reveal the presence of numerous bright dots in the cortical neuropile with a gradient of density from the ventricular surface to the surface of the brain. ( D ) Coronal sections of E14.5 brain are labeled with SHH-Nter antibodies ( D1 ) or only with the secondary antibodies ( D2 ). Confocal Z stacks of 48 images (△z=0.2 µm) reveal a punctiform signal all over the cortical neuropile and in some cells in the cortical plate ( D1 ), whereas no signal is observed in sections immunolabeled only with the secondary fluorescent antibody ( D2 ). CP, choroid plexus; Cx, cortex; Sp, septum; V3, third ventricle; ZLI, zona intra-thalamica. Scale bar: 250 µm ( A, B ), 100 µm ( C, D ).

Article Snippet: Primary antibodies were goat anti SHH-Nter (1:100, R&D Systems AF464, RRID: AB_355373 ), goat anti Netrin G1a (NG1a) (1:100, R&D Systems AF1166, RRID: AB_2154822 ), rabbit anti TBR1 (1:1000, Abcam ab31940, RRID: AB_2200219 ), rabbit anti TBR2 (1:1000, Abcam ab23345, RRID: AB_778267 ), rabbit anti PAX6 (1:100, clone poly19013, Covance PRB-278P, RRID: AB_291612 ), rabbit anti GSH2 (1:2000, Millipore ABN162, RRID: AB_11214376 ), chicken anti MAP2 (1:500, Novus, NB30213), mouse anti SMI-32 (1:500, Covance SMI-32R, RRID: AB_509997 ), and rat CTIP2 (1:1000, Abcam ab18465, RRID: AB_2064130 ).

Techniques: Immunostaining, Labeling, Immunolabeling

Journal: Journal of Clinical Medicine

Article Title: Reduced Salivary Gustin and Statherin in Long-COVID Cohort with Impaired Bitter Taste

doi: 10.3390/jcm13226816

Figure Lengend Snippet: ( A ) Epithelial cells in saliva stained with AO/PI. Majority of the epithelial cells are viable as indicated by AO+ staining. ( B ) A representative image of cell in saliva staining positive for SHH and ( C ) occludin.

Article Snippet: Immunofluorescence was performed using primary antibodies against pan cytokeratin (1:100; pan-Cytokeratin Antibody (AE1/AE3): sc-81714, Santa Cruz Biotechnology, Inc.; Dallas, TX, USA), SHH (Catalog #: AF464, R&D Systems, Minneapolis, MN, USA), and occludin (1:250: sc-133256, Santa Cruz Biotechnology, Inc.; Dallas, TX, USA).

Techniques: Staining

Journal: Journal of developmental biology

Article Title: A Residual N-Terminal Peptide Enhances Signaling of Depalmitoylated Hedgehog to the Patched Receptor.

doi: 10.3390/jdb12020011

Figure Lengend Snippet: Figure 2. Reverse-phase HPLC reveals the decreased hydrophobicity of ShhC released in the presence of serum or of pharmacological cholesterol acceptors. Goat-α-Shh antibodies (R&D Systems, AF464) were used for all blots to detect full-length unprocessed Shh (decreased electrophoretic mobility band, top) and N-truncated proteins that were solubilized from the cellular precursor (increased electrophoretic mobility band, bottom). Lower fraction numbers (fr#) indicate more hydrophilic (delipidated) proteins and higher fraction numbers indicate more lipophilic (lipidated) proteins. (A–D) RP-HPLC calibration. Consistent with its dual lipidation, R&D 8908-SH positive control proteins elute predominantly in the late fraction #37 (black arrowhead). Artificial monolipidated cellular ShhN elutes in fractions #27–29 (red arrowhead) and monolipidated cellular C25SShh (this artificial variant has the cysteine palmitate acceptor replaced with a non-accepting serine) elutes predominantly in fraction #32 (white arrowhead). Overexpressed soluble C25SShhN, another engi- neered control protein lacking both lipids, elutes in fractions #26–28 from the C4 column (green arrowhead). N-terminal Shh peptides in the schematics are labeled in orange. (E) Overexpressed cellular Shh elutes predominantly in fraction #37 (black arrowhead); a small fraction that probably did not undergo Hhat-dependent N-terminal palmitoylation elutes in fraction #33 (white arrowhead). (F) ShhC, solubilized by Disp and Scube2 from its dually lipidated cellular precursor (E), also eluted in fractions #32–34, showing that it retained the C-cholesterol moiety but not the N-palmitate after its release (white arrowhead). (G) Consistent with this, the artificially produced soluble control C25SShhC, blocked in its ability to undergo N-palmitoylation during biosynthesis, also eluted in fractions #32–34 (white arrowhead). The increased electrophoretic mobility of the protein indicated that its N-terminus was also processed. (H,I) Similar hydrophobicity of ShhC and C25SShhC was expressed in the presence of 600 µg/mL of the pharmacological cholesterol acceptor CD. Note that the electrophoretic mobility of the most soluble ShhC is again increased (lower band), consistent with proteolytic processing of the palmitoylated N-terminal peptide during Disp- and Scube2-mediated ShhC release [32,36].

Article Snippet: Goat-α-Shh antibodies (R&D Systems, AF464) were used for all blots to detect full-length unprocessed Shh (decreased el ctrophoretic mobility and, top) and N-truncated pro ins that were solubilized from the cellular precurso (increased electrophoretic mobility band, bottom).

Techniques: Positive Control, Variant Assay, Control, Labeling, Produced

Journal: Journal of developmental biology

Article Title: A Residual N-Terminal Peptide Enhances Signaling of Depalmitoylated Hedgehog to the Patched Receptor.

doi: 10.3390/jdb12020011

Figure Lengend Snippet: Figure 4. A minimal N-terminal amino acid sequence contributes to Shh signaling in vitro. (A) The N-terminal Shh peptide, including the Cardin–Weintraub (CW) motif (green) is shown. A G-to-R exchange (shown in blue) just upstream of the CW site renders G32RShh susceptible to furin cleavage (inset, right lane, red arrowhead). PCSK7 cleaves the wild-type Shh peptide at the same site (inset, and red arrowhead). (B) qPCR confirmed that ShhC and C25AShhC increased the transcription of Ptch1, Gli1, and Alp1 in C3H10T1/2 cells to a similar extent. Target gene transcription was much less induced by furin- or PCSK7-cleaved proteins, indicating that processing rendered them inactive. (C) Cryo-EM derived structures (pdb: 6e1h) reveal interactions between the palmitoylated Shh N-peptide (white backbone, nitrogens stained blue, oxygens stained red, palmitate stained green) and Ptch residues (stained yellow) [22]. (D) Shh and mutant proteins lacking the N-terminal C25 to prevent palmitoylation, and their consecutively N-truncated counterparts were expressed, pulled down with heparin, and immunoblotted. All proteins were expressed at similar levels, as indicated by polyclonal α-Shh reactivity. Bottom: C3H10T1/2 osteoblast progenitor cells were incubated with similar amounts of ShhC, C25SShhC, and their truncated variants, and relative increases in Alp1 activity were determined as biological readouts. Media obtained from mock-transfected Bosc23 cells was used as a negative control, and ShhC-conditioned media as a positive control. Inhibition of ShhC

Article Snippet: Goat-α-Shh antibodies (R&D Systems, AF464) were used for all blots to detect full-length unprocessed Shh (decreased el ctrophoretic mobility and, top) and N-truncated pro ins that were solubilized from the cellular precurso (increased electrophoretic mobility band, bottom).

Techniques: Sequencing, In Vitro, Cryo-EM Sample Prep, Derivative Assay, Staining, Mutagenesis, Incubation, Activity Assay, Transfection, Negative Control, Positive Control, Inhibition