Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: NGF-induced PC12 cell differentiation toward sympathetic neurons and expressions of GAP-43 and TH. (A) Immunofluorescent stains of GAP-43 (red, upper row) and TH (red, lower row). Nuclei were stained blue by DAPI. Note that NGF induced significant neurite outgrowth. Scale bar, 10 μm. (B,C) Real-time PCR and western blotting results of GAP-43 and TH expressions in PC12 cells treated or untreated with NGF. ∗∗∗ p < 0.001 vs. respective control. ### p < 0.001 vs. respective control; n = 3 for each value.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Cell Differentiation, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: The suppressing effects of ivabradine (IVA) on the mRNA and protein expressions of HCN channel isoforms and GAP-43 and the neurite outgrowth. (A–D) Real-time PCR and western blotting results of the mRNA and protein expression levels respectively for HCN1-4 isoforms in NGF-treated PC12 cells relative to GADPH level. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. NGF treatment alone in the mRNA level. # p < 0.05, ## p < 0.01 vs. NGF treatment alone in the protein level. (E) Representative western blotting bands ( upper ) and the mRNA and protein semiquantitative values ( lower ) of GAP-43 in NGF-treated PC12 cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. NGF treatment alone in the mRNA level. # p < 0.05, ### p < 0.001 vs. NGF treatment alone in the protein level. (F) Immunofluorescent stains of GAP-43 protein (red), displaying that NGF induced, while ivabradine inhibited, the neurite outgrowth in differentiating PC12 cells. DAPI was used for cell nuclei staining (blue). Scale bar, 10 μm. (G,H) Quantitative morphological parameters of neurite outgrowth including the total neurite length and the maximal neurite length (longest process per cell). Results are calculated from three independent experiments and presented as mean ± SEM, 96 < n < 213, ∗ p < 0.05, ∗∗ p < 0.01 vs. NGF treatment alone.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: Knocking-down HCN2 and HCN4 expression with siRNAs inhibited GAP-43 expression and neurite outgrowth in PC12 cells. NGF-treated PC12 cells were transiently transfected with the negative control siRNA (NC-siRNA) or with the siRNA respectively for HCN1, HCN2, HCN3 or HCN4 using Lipofectamine ® RNAiMAX Reagent for 48 h before cells were harvested for detection. (A–D) The mRNA and protein expressions of HCN1-4 isoforms assayed by qPCR and western blotting in comparison to GADPH in NGF-treated PC12 cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. negative control siRNA (N.C.) in the mRNA level. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. N.C. in the protein level. (E) Representative western blotting results ( upper ) and the densitometric analysis of GAP-43 mRNA and protein expressions ( lower ) ( n = 3–6 independent experiments). ∗ p < 0.05, ∗∗∗ p < 0.001 vs. N.C. in the mRNA level. ### p < 0.001 vs. N.C. in the protein level. (F) Immunofluorescent stains of GAP-43 in PC12 cells transiently transfected with siRNA targeting HCN2 or HCN4. Scale bar, 10 μm. (G,H) Quantification of neurite morphological parameters (total length of neurite outgrowth and longest process per cell). Results are calculated from three independent experiments and presented as mean ± SEM, 50 < n < 84, ∗ p < 0.05, ∗∗ p < 0.01 vs. N.C.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Expressing, Transfection, Negative Control, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: Overexpression of HCN2 and HCN4 enhanced GAP-43 expression and neurite outgrowth in PC12 cells. Results were obtained 48 h after cell transfection with either empty plasmid pcDNA3.0/vector or pcDNA3.0/HCN2 or pcDNA3.0/HCN4. (A,B) Representative western blots ( upper ) and semiquantitative values ( lower ) of the mRNA and protein levels of HCN2 and HCN4 isoforms. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. control (pcDNA3.0/vector) in the mRNA level. # p < 0.05 vs. control (pcDNA3.0/vector) in the protein level, n = 4−6 independent experiments. (C) qPCR and western blotting results showing the effects of HCN2 or HCN4 overexpression on the mRNA and protein expressions of GAP-43. ∗∗∗ p < 0.001 vs. empty plasmid in the mRNA level. ### p < 0.001 vs. empty plasmid in the protein level, n = 4−6 independent experiments. (D) Immunofluorescent stains of GAP-43 (red) of PC12 cells transiently transfected respectively with pcDNA3.0/vector, pcDNA3.0/HCN2 or pcDNA3.0/HCN4 plasmids. Scale bar, 10 μm. (E,F) Quantification of neurite morphological parameters including total neurite outgrowth and longest process per cell. Results are calculated from three independent experiments and presented as mean ± SEM, 50 < n < 90, ∗ p < 0.05 vs. empty vector.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot

Journal: The Journal of Neuroscience

Article Title: Intraneural Injection of ATP Stimulates Regeneration of Primary Sensory Axons in the Spinal Cord

doi: 10.1523/JNEUROSCI.1660-17.2017

Figure Lengend Snippet: Antibodies used

Article Snippet: GAP43 , 1:10,000 , Atlas Antibodies , HPA015600 , AB_1234436.

Techniques:

Journal: The Journal of Cell Biology

Article Title: Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex

doi: 10.1083/jcb.200507061

Figure Lengend Snippet: Ral regulates GAP-43 phosphorylation. (A) COS cells were transfected with the indicated myc-tagged Ral constructs and cGAP-43. Levels of phospho-cGAP-43 in cell extracts were determined by immunoblotting with an anti–phospho-GAP-43 antibody. A representative blot is shown. Inactive Ral causes a significant decrease of cGAP-43 phosphorylation (top band, arrow). (B) Quantitative analysis of phosphorylated cGAP-43 in COS cells transfected with cGAP-43 and the indicated Ral constructs. Shown here are data from four independent experiments (means ± SEM; *, P < 0.01). (C) Cortical neurons were nucleofected with the indicated constructs and lysed after overnight expression. Levels of total and phosphoendogenous GAP-43 were then assessed by immunoblotting. The fourth, fifth, and sixth lanes were derived from the same blot. (D) Quantitative analysis of endogenous GAP-43 phosphorylation in cortical neurons after nucleofection with the indicated Ral mutant isoforms. Data were derived from four independent experiments (means ± SEM; **, P < 0.005).

Article Snippet: Antibodies used were mouse anti-RalA, rabbit anti-RalB (BD Biosciences), mouse anti–GAP-43 (Autogen Bioclear), rabbit anti–phospho(Ser41)-GAP-43 (Zymed Laboratories), rat anti–α-tubulin (Harlan), rat anti-HA (Roche), mouse anti-myc (clone 9E10), rabbit anti-myc (Research Diagnostics, Inc.), mouse anti–βIII tubulin (Sigma-Aldrich), and rabbit anti-p38 (Santa Cruz Biotechnology, Inc.).

Techniques: Transfection, Construct, Western Blot, Expressing, Derivative Assay, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex

doi: 10.1083/jcb.200507061

Figure Lengend Snippet: GAP-43 acts downstream of Ral. (A) Constitutively active Ral increases branching in neurons plated on laminin (top middle and right) compared with EGFP-F–expressing cells (top left). This effect is reduced by coexpression of cGAP-43(S42A) (middle and bottom). Shown here are cells stained with anti-myc antibody to detect mutant Ral and anti–cGAP-43. Bar, 100 μm. (B) Quantitative analysis of branching in cells injected with single Ral isoforms or with Ral and cGAP-43(S42A) (means ± SEM: GFP, 3.30 ± 0.18; RalA72L, 8.43 ± 0.97; RalA72L + cGAP-43(S42A), 3.55 ± 0.44; RalB23V, 8.88 ± 0.85; RalB23V + cGAP-43(S42A), 4.29 ± 0.41; *, P < 0.02; **, P < 0.0001).

Article Snippet: Antibodies used were mouse anti-RalA, rabbit anti-RalB (BD Biosciences), mouse anti–GAP-43 (Autogen Bioclear), rabbit anti–phospho(Ser41)-GAP-43 (Zymed Laboratories), rat anti–α-tubulin (Harlan), rat anti-HA (Roche), mouse anti-myc (clone 9E10), rabbit anti-myc (Research Diagnostics, Inc.), mouse anti–βIII tubulin (Sigma-Aldrich), and rabbit anti-p38 (Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Staining, Mutagenesis, Injection

Journal: The Journal of Cell Biology

Article Title: Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex

doi: 10.1083/jcb.200507061

Figure Lengend Snippet: GAP-43 acts downstream of Ral. (A) A phosphomimetic GAP-43 protein, cGAP-43(S42D), restores branching in neurons plated on laminin and expressing dominant-negative Ral. Shown here are neurons expressing EGFP-F (top left), RalA28N (top middle), or RalB28N (bottom left) and RalA28N or RalB28N and cGAP-43(S42D) (right). Cells are stained with an anti-myc antibody to detect mutant Ral and an anti–cGAP-43 antibody. Bar, 50 μm. (B) Quantitative analysis of branching in cells plated on laminin and injected with single Ral isoforms or with Ral and cGAP-43(S42D) (means ± SEM: GFP, 3.47 ± 0.37; RalA28N, 1.74 ± 0.52; RalA28N + cGAP-43(S42D), 2.42 ± 0.31; RalB28N, 1.56 ± 0.41; RalB28N + cGAP-43(S42D), 3.72 ± 0.35; *, P < 0.04; **, P < 0.001). (C) SCG neurons were plated on polyornithine, microinjected, and left to express the indicated proteins for 5 h before laminin addition. Expression of dominant-negative Ral decreases branching compared with EGFP-F–expressing cells (top, compare middle and right with left). Branching is restored in cells coexpressing cGAP-43(S42D) (middle and bottom rows). Bar, 100 μm. (D) Quantitative analysis of branching in neurons initially plated on polyornithine and injected with single Ral isoforms or with Ral and cGAP-43(S42A) before laminin addition (means ± SEM: GFP, 2.64 ± 0.35; RalA28N, 1.05 ± 0.32; RalA28N + cGAP-43(S42D), 1.82 ± 0.40; RalB28N, 1.74 ± 0.30; RalB28N + cGAP-43(S42D), 2.73 ± 0.45; **, P < 0.01).

Article Snippet: Antibodies used were mouse anti-RalA, rabbit anti-RalB (BD Biosciences), mouse anti–GAP-43 (Autogen Bioclear), rabbit anti–phospho(Ser41)-GAP-43 (Zymed Laboratories), rat anti–α-tubulin (Harlan), rat anti-HA (Roche), mouse anti-myc (clone 9E10), rabbit anti-myc (Research Diagnostics, Inc.), mouse anti–βIII tubulin (Sigma-Aldrich), and rabbit anti-p38 (Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Dominant Negative Mutation, Staining, Mutagenesis, Injection

Journal: Scientific Reports

Article Title: The BEACH Protein LRBA Promotes the Localization of the Heterotrimeric G-protein G olf to Olfactory Cilia

doi: 10.1038/s41598-017-08543-4

Figure Lengend Snippet: Histological and subcellular distribution of LRBA in olfactory and vomeronasal tissues. ( A ) LacZ enzyme histochemistry of Lrba/gt gene-trap mice (age, 3 weeks) demonstrates expression of the LRBA gene throughout the OE (top). Larger magnification (middle) shows that the staining is predominantly localized to the OSNs. Respiratory epithelium (RE, bottom) is also stained, though weaker than OE. ( B ) β-galactosidase staining was also seen in VNO neuron somata and the microvillar layer of Lrba/gt mice (top, middle), and confirmed by LRBA-IF of adult WT tissue (bottom; SE, sensory epithelium; L, lumen). Scale bars in A , B : overviews 100 µm, close-ups 25 µm. ( C ) LacZ staining of the olfactory bulb required longer incubation (16 h) than of the OE and VNO (2 h). Scale bar in C : 100 µm. ( D – K ) Triple immunostaining for LRBA ( D , H ), OMP ( E , I ), GAP43 ( F , J ) and merged images ( G , K ) in different regions of the adult mouse OE of low ( D – G ) or high ( H–K ) thickness. LRBA-immunoreactivity (ir) is found in the apical somatic cytoplasm and in dendrites as well as dendritic knobs of virtually all OMP-ir mature OSNs. Large and small arrows point to select LRBA-/OMP-ir cell bodies/dendrites and dendritic knobs, respectively, but LRBA is lacking in the OMP-ir ciliary layer (small arrowheads in D – G ). GAP43-ir immature neurons contain no (large arrowheads in D – G ) or only small amounts (double arrows in D – K ) of LRBA-immunoreactivity in the apical cytoplasm of their cell bodies and in dendritic knobs. Scale bar in K for D – K : 10 µm. OE strata are designated in panels G and K: cl, cilial layer; scl, sustentacular cell layer; osnl, OSN layer.

Article Snippet: Antibodies used were rabbit anti-mLRBA-B (1:100), goat anti-OMP (1:300; Wako) as a marker for mature OSNs , and mouse anti-GAP43 (1:300; MerckMillipore) as a marker for immature OSNs , .

Techniques: Expressing, Staining, Incubation, Triple Immunostaining