mouse anti elavl4  (Proteintech)

Journal: International journal of molecular sciences

Article Title: The Antidepressant Effect of Resveratrol Is Related to Neuroplasticity Mediated by the ELAVL4- Bdnf mRNA Pathway.

doi: 10.3390/ijms26031113

Figure Lengend Snippet: Figure 3. ELAVL4 as a key protein in the antidepressant effect of resveratrol: (A) Venn diagram of hippocampal proteomic showing downregulated DEPs (Cums vs. Con) and upregulated DEPs (Res

Article Snippet: The samples were then incubated in 5% BSA (4240GR100, BioFroxx, Bavaria, Germany) for 1 h and subsequently treated with primary mouse anti-ELAVL4 antibody (dilution 1:1000, 67835-1-lg, Proteintech, Chicago, IL, USA), and rabbit anti-BDNF antibody (dilution 1:1000, ER130915, Huabio, Hangzhou, China) overnight at 4 ◦C.

Techniques:

Journal: International journal of molecular sciences

Article Title: The Antidepressant Effect of Resveratrol Is Related to Neuroplasticity Mediated by the ELAVL4- Bdnf mRNA Pathway.

doi: 10.3390/ijms26031113

Figure Lengend Snippet: Figure 4. Bdnf as a potential downstream target of ELAVL4: (A) Venn diagram showing the overlap between ELAVL4 target mRNAs and neuroplasticity-related genes. (B,C) Interaction network of the top 100 interactions of ELAVL4 or Bdnf mRNA. (D) AlphaFold3-predicted structural interaction between ELAVL4 protein and Bdnf mRNA. (E) Relative Bdnf expression/Gapdh mRNA expres- sion among the four groups. (F,G) Relative expression of BDNF normalized to GAPDH (N = 3). (H–M) Representative immunofluorescence images showing ELAVL4 and BDNF expression in the DG and CA3 region of the hippocampus (N = 3). Scale bars represent 75 µm. Statistical analyses are performed using one-way ANOVA with Tukey’s post hoc test. (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: The samples were then incubated in 5% BSA (4240GR100, BioFroxx, Bavaria, Germany) for 1 h and subsequently treated with primary mouse anti-ELAVL4 antibody (dilution 1:1000, 67835-1-lg, Proteintech, Chicago, IL, USA), and rabbit anti-BDNF antibody (dilution 1:1000, ER130915, Huabio, Hangzhou, China) overnight at 4 ◦C.

Techniques: Expressing, Immunofluorescence

Journal: iScience

Article Title: Placode and neural crest origins of congenital deafness in mouse models of Waardenburg-Shah syndrome

doi: 10.1016/j.isci.2024.111680

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-HuD (Elavl4) antibody (E-1) , Santa Cruz Biotechnology , Cat# sc-28299; RRID: AB_627765.

Techniques: Recombinant, Purification, Software

Journal: Cell Reports

Article Title: Mutant FUS and ELAVL4 (HuD) Aberrant Crosstalk in Amyotrophic Lateral Sclerosis

doi: 10.1016/j.celrep.2019.05.085

Figure Lengend Snippet: FUS P525L Binds and Regulates the ELAVL4 3′ UTR (A) Snapshot of the IGV window showing the mapping of PAR-CLIP reads and transitions on the ELAVL4 3′UTR. (B) Upper panel, schematic representation of the enhanced piggyBac transposable vector for doxycycline conditional expression of RFP-FUS WT (epB-Puro-TT-RFP-FUS WT ), RFP-FUS P525L (epB-Puro-TT-RFP-FUS P525L ), and RFP control (epB-Puro-TT-RFP). F, FLAG tag; pA, polyadenylation signal; PuroR, puromycin resistance gene; T2A, self-cleavage peptide; rtTA, TET transactivator protein gene; Pubc, human ubiquitin C constitutive promoter; TRE, TET responsive element; Dox, doxycycline. Triangles represent terminal repeats of the transposon. Lower panels, HeLa cells expressing RFP, RFP-FUS WT , RFP-FUS P525L (red) and stained with Hoechst (blue) for nuclei. Scale bars, 20 μm. (C) Luciferase assay on selected target 3′ UTRs in HeLa cells expressing RFP, RFP-FUS WT , and RFP-FUS P525L . (D) Measurement of the RNA levels of the Luciferase-ELAVL4-3′UTR by real time qRT-PCR (relative expression compared to RFP-transfected cells). Histogram bars represent the average of 3 independent experiments and error bars indicate the SD (Student’s t test; paired; two tails; ∗ p < 0.05; n.s., p > 0.05). (E) ELAVL4 protein quantification by western blot in FUS WT and FUS P525L FACS-purified MNs at two different time points of differentiation (see Figure S1 A). (F) ELAVL4 RNA levels were measured by real time qRT-PCR. Expression levels in FUS P525L MNs are shown as relative to the isogenic FUS WT control, set to a value of 1 (dashed bar). Histogram bars represent the average of 3 independent experiments and error bars indicate the SD (Student’s t test; paired; two tails; ∗ p < 0.05; ∗∗ p < 0.01). See also Figure S3 .

Article Snippet: Mouse monoclonal anti-ELAVL4 antibody , Santa Cruz Biotechnology , Cat#sc-48421; RRID: AB_627766.

Techniques: Plasmid Preparation, Expressing, Control, FLAG-tag, Ubiquitin Proteomics, Staining, Luciferase, Quantitative RT-PCR, Transfection, Western Blot, Purification

Journal: Cell Reports

Article Title: Mutant FUS and ELAVL4 (HuD) Aberrant Crosstalk in Amyotrophic Lateral Sclerosis

doi: 10.1016/j.celrep.2019.05.085

Figure Lengend Snippet: ELAVL4 Forms Cytoplasmic Speckles and Interacts with FUS P525L in the Cytoplasm (A) Schematic representation of the enhanced piggyBac transposable vector for doxycycline conditional expression of GFP-ELAVL4 (epB-Bsd-TT-GFP-ELAVL4). F, FLAG tag; H, HA tag; pA, polyadenylation signal; BsdR, blasticidin resistance gene; T2A, self-cleavage peptide; rtTA, TET transactivator protein gene; Pubc, human ubiquitin C constitutive promoter; TRE, TET responsive element; Dox, doxycycline. Triangles represent terminal repeats of the transposon. (B) Confocal images of HeLa cells stably transduced with the epB-Bsd-TT-GFP-ELAVL4 vector and induced with doxycycline for 24 h. Hoechst (blue) marks nuclei. Scale bar, 10 μm. (C) Confocal images of HeLa cells stably transduced with the epB-Bsd-TT-GFP-ELAVL4 and epB-Puro-TT-RFP-FUS WT (top panels) or epB-Puro-TT-RFP-FUS P525L (bottom panels) vectors and induced with doxycycline for 24 h. In the MERGE panels DAPI (blue) marks nuclei. Scale bar, 20 μm. (D) Representative images acquired with the confocal microscope (left) and with the BDB microscope (right) of a HeLa cell expressing GFP-ELAVL4 and RFP-FUS P525L . Scale bar, 10 μm. (E) Bar plot of the Brillouin frequency shift in cytoplasmic compartments of HeLa cells expressing GFP-ELAVL4, RFP-FUS P525L , or both. Grey bars are relative to regions of the cytoplasm with high RFP and/or GFP signal. White bars refer to the rest of the cytoplasm. Histogram bars represent the average (6, 5, and 12 cells, respectively; two independent experiments) and error bars indicate the SD (p values from Student’s t test; paired; two tails). (F) Co-immunoprecipitation of RFP-FUS P525L and GFP-ELAVL4 from cytoplasmic extracts of HeLa cells treated as in (C). Immunoprecipitation was performed using an anti-FUS antibody. Samples were untreated or treated with RNaseA before immunoprecipitation. RFP-FUS P525L and GFP-ELAVL4 bands were revealed with an anti-FLAG antibody. (G) Co-immunoprecipitation of endogenous FUS P525L and ELAVL4 in cytoplasmic extract of motor neuron by using a FUS antibody and revealed by western blot with anti-FUS and anti-ELAVL4 antibodies. See also Figure S4 .

Article Snippet: Mouse monoclonal anti-ELAVL4 antibody , Santa Cruz Biotechnology , Cat#sc-48421; RRID: AB_627766.

Techniques: Plasmid Preparation, Expressing, FLAG-tag, Ubiquitin Proteomics, Stable Transfection, Transduction, Microscopy, Immunoprecipitation, Western Blot

Journal: Cell Reports

Article Title: Mutant FUS and ELAVL4 (HuD) Aberrant Crosstalk in Amyotrophic Lateral Sclerosis

doi: 10.1016/j.celrep.2019.05.085

Figure Lengend Snippet: ELAVL4 and FUS Localization in Human iPSC-Derived Motor Neurons in Normal or Stress Conditions (A) Immunostaining for FUS (red) and ELAVL4 (green) in FUS WT and FUS P525L MNs at 7 days after FACS purification in untreated control conditions. In the MERGE panels, DAPI (blue) stains nuclei. (A’) Immunostaining for ELAVL4 (green), TIAR (red), and with DAPI (blue) in cells treated as in (A). Single channels are shown in Figure S6 . (B) Immunostaining for FUS (red) and ELAVL4 (green) in FUS WT and FUS P525L MNs at 7 days after FACS purification, cultured in presence of 0.5 mM sodium arsenite for 90 min. In the MERGE panels, DAPI (blue) stains nuclei. (B’) Immunostaining for ELAVL4 (green), TIAR (red), and with DAPI (blue) in cells treated as in (B). Single channels are shown in Figure S6 . Scale bar, 10 μm. (C) Scatter dot plot representing the quantitative analysis, in one representative experiment, of the volumes (left) and mean intensity (total signal intensity normalized for the volume) of ELAVL4 signal (right) of the ELAVL4-positive speckles in FUS WT (n = 23) and FUS P525L (n = 38) MNs as shown in (A). (D) Scatter dot plot representing the quantitative analysis of the volumes (left) and mean intensity (total signal intensity normalized for the volume), in one representative experiment, of ELAVL4 signal (right) of the ELAVL4-positive SGs in FUS WT (n = 23) and FUS P525L (n = 30) MNs as shown in (B). In all graphs the bars indicate the median with interquartile range. p values from Student’s t test (unpaired; two tails) are indicated. See also , , and .

Article Snippet: Mouse monoclonal anti-ELAVL4 antibody , Santa Cruz Biotechnology , Cat#sc-48421; RRID: AB_627766.

Techniques: Derivative Assay, Immunostaining, Purification, Control, Cell Culture

Journal: Cell Reports

Article Title: Mutant FUS and ELAVL4 (HuD) Aberrant Crosstalk in Amyotrophic Lateral Sclerosis

doi: 10.1016/j.celrep.2019.05.085

Figure Lengend Snippet: ELAVL4 Immunostaining Analysis in FUS Patients’ Specimens Immunofluorescence showing spinal cord motor neurons of ALS patients harboring the indicated FUS mutations and control. Sections were stained with anti-FUS (red) and anti-ELAVL4 (green) antibodies and counterstained with Hoechst. Scale bar, 20 μm. Characteristics of patients and controls are reported in .

Article Snippet: Mouse monoclonal anti-ELAVL4 antibody , Santa Cruz Biotechnology , Cat#sc-48421; RRID: AB_627766.

Techniques: Immunostaining, Immunofluorescence, Control, Staining

Journal: Cell Reports

Article Title: Mutant FUS and ELAVL4 (HuD) Aberrant Crosstalk in Amyotrophic Lateral Sclerosis

doi: 10.1016/j.celrep.2019.05.085

Figure Lengend Snippet: ELAVL4 Immunohistochemistry Analysis in Sporadic ALS Patients’ Specimens Immunohistochemistry on spinal cord samples from sporadic ALS patients. For double immunohistochemistry, sections were labeled with anti-ELAVL4 (blue) and anti-pTDP-43 (pS409/410) (red). Scale bar: 20 μm. Arrows indicate pTDP-43 and ELAVL4 double positive cytoplasmic inclusions. Characteristics of patients and controls are reported in . See also Figure S8 .

Article Snippet: Mouse monoclonal anti-ELAVL4 antibody , Santa Cruz Biotechnology , Cat#sc-48421; RRID: AB_627766.

Techniques: Immunohistochemistry, Labeling

Journal: Cell Reports

Article Title: Mutant FUS and ELAVL4 (HuD) Aberrant Crosstalk in Amyotrophic Lateral Sclerosis

doi: 10.1016/j.celrep.2019.05.085

Figure Lengend Snippet: Model and Expression of FUS Pathological Inclusion Components in Human (A) Model of the aberrant crosstalk between mutant FUS and ELAVL4 in ALS. (i) In normal conditions FUS (red) is mainly localized in the nucleus. ELAVL4 (green) is localized both in the nucleus and in the cytoplasm, where it has a diffuse localization punctuated by speckles. Human iPSC-derived FUS WT MNs would be representative of this situation. (ii) ALS mutations in the nuclear localization signal cause FUS partial de-localization in the cytoplasm. Mutant FUS binds ELAVL4 3′UTR. Moreover, as a consequence of FUS nuclear depletion, miR-375 levels decrease. These events gradually cause an increase of ELAVL4 protein levels. Human iPSC-derived FUS P525L MNs would be representative of this situation. (iii) Over time, ELAVL4 protein levels are increasingly augmented, forming bigger speckles in the cytoplasm. It is possible that protein-protein interaction with ELAVL4 results in further mutant FUS accumulation in the cytoplasm. This, in turn, would further increase ELAVL4 levels by the above-mentioned mechanisms. (iv) Increased levels of both ELAVL4 and mutant FUS in the cytoplasm and their interaction might lead to the formation of speckles containing both proteins, independently from cellular stress. This situation cannot be observed in human iPSC-derived MNs, which correspond to “young” MNs. However, mutant FUS/ELAVL4 speckles are formed in the cytoplasm of HeLa cells overexpressing both proteins. (v) In presence of cellular stress, e.g., oxidative stress, cytoplasmic mutant FUS and ELAVL4 proteins co-localize in stress granules. We observe this phenomenon in both transgenic HeLa cells and human iPSC-derived MNs. (vi) Alteration of granules/speckles biomechanical properties due to mutant FUS and/or increased ELAVL4 would lead over time to the formation of ALS pathological inclusions, in which mutant FUS, ELAVL4, and SG markers can be detected. As previously proposed ( Bentmann et al., 2013 ), three modes of pathological inclusions formation are possible: SG-dependent, aggregation-dependent, aggregation- and SG-dependent. (B) Heatmap representing gene expression levels in different human tissues from the GTEx Portal ( https://www.gtexportal.org/home/ ). Genes that are known components of FUS pathological inclusions ( Blokhuis et al., 2013 , Blokhuis et al., 2016 ) are reported. The ubiquitous ELAVL family member ELAVL1 ( HuR ) is also shown. Expression values are shown in TPM (transcripts per million), calculated from a gene model with isoforms collapsed to a single gene. See also Figure S9 .

Article Snippet: Mouse monoclonal anti-ELAVL4 antibody , Santa Cruz Biotechnology , Cat#sc-48421; RRID: AB_627766.

Techniques: Expressing, Mutagenesis, Derivative Assay, Transgenic Assay, Gene Expression

Journal: Cell Reports

Article Title: Mutant FUS and ELAVL4 (HuD) Aberrant Crosstalk in Amyotrophic Lateral Sclerosis

doi: 10.1016/j.celrep.2019.05.085

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-ELAVL4 antibody , Santa Cruz Biotechnology , Cat#sc-48421; RRID: AB_627766.

Techniques: Recombinant, Luciferase, Plasmid Preparation, SYBR Green Assay, Software, Imaging, Sequencing, Gene Expression, Expressing