Journal: Molecular Neurodegeneration

Article Title: Sensory-motor deficits and neurofilament disorganization in gigaxonin-null mice

doi: 10.1186/1750-1326-6-25

Figure Lengend Snippet: Increased abundance of NF subunits in the three GAN models . The relative increase of protein content was obtained by comparing the mean abundance in each of the three GAN models (KO1 = our GAN ex3-5 ; KO2 = GAN YY ; KO3 = GAN ex1 ) with WT mice (n = 3 mice per genotype, except n = 2 for 48 week-old KO2). (A) Expression levels were quantified in the brain, the lumbar section of spinal cord (Sc-L) and sciatic nerves (SN) by immunoblotting using anti-NFL, NFM and NFH antibodies and normalization with GAPDH antibody. (B) The relative abundance of the gigaxonin's partners MAP1B, MAP1S and TBCB was quantified using the corresponding antibodies, with a similar approach. (Mann-Whitney test, *, p < 0.05; bars represent standard deviation). The immunoblots corresponding to the abundance of the NF subunits and the gigaxonin's partners in brain of 48 week-old GAN models are represented in (C).

Article Snippet: Antibodies: mouse anti-gigaxonin [ ] (N12, 1:150), rabbit anti-MAP1B-LC and anti-MAP1S-HC (kind gift from Pr Propst, 1:500), mouse anti-TBCB (AbnovaH00001155-A01, 1:250), mouse anti-NFL (Millipore MAP1615, 1:250), mouse anti- NFH (Millipore MAB5266, 1:200), mouse anti-NFM (Millipore MAB5254, 1:500), mouse anti-ßactin (Abcam 8226, 1:1000), mouse anti-GAPDH (Ambion 4300, 1:4000).

Techniques: Expressing, Western Blot, MANN-WHITNEY, Standard Deviation

Journal: Life Science Alliance

Article Title: Multi-omics profiling identifies a deregulated FUS-MAP1B axis in ALS/FTD–associated UBQLN2 mutants

doi: 10.26508/lsa.202101327

Figure Lengend Snippet: (A) Common candidates of the transcriptome and proteome analyses. Only candidates which were commonly regulated in at least four of the eight omics data sets were included. Blue and red indicate significantly decreased and increased mRNA and protein abundance, respectively. (B) Gene expression changes of MAP1B in engineered HeLa and patient-derived LCL cells measured by RT-PCR. Error bars represent standard error of the mean of three biological replicates (** P < 0.01, *** P < 0.001, t test). (C) Schematic representation of MAP1B. ABD, actin-binding domain. MBD, microtubule-binding domain. (D) Protein abundance changes of MAP1B full-length and light chain (MAP1B-LC1) from engineered HeLa and patient-derived LCL cells. (E) Representative images of patient-derived LCL cells fixed and stained with a MAP1B antibody and DAPI. Scale bar: 5 μm.

Article Snippet: Rabbit anti-MAP1B , Sigma-Aldrich , HPA022275.

Techniques: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Staining

Journal: Life Science Alliance

Article Title: Multi-omics profiling identifies a deregulated FUS-MAP1B axis in ALS/FTD–associated UBQLN2 mutants

doi: 10.26508/lsa.202101327

Figure Lengend Snippet: (A) Immunoblot of UBQLN2 WT and KO HeLa cells. (B) Representative images of UBQLN2 WT and KO cells fixed and immunostained for UBQLN2 and MAP1B. Scale bar: 20 μm. (C) Immunoblot analysis of UBQLN2 WT and KO cells transfected with HA-UBQLN2 or left untreated (MOCK; only Lipofectamine). (C, D) Quantification of MAP1B protein levels from (C). Data represent mean ± SD. Statistical analysis (n = 3) of the MAP1B protein/control protein ratio was performed using one-way ANOVA followed by Tukey’s post hoc test. n.s., not significant. (E) Ubqln2 knockdown in primary hippocampal rat neurons transduced with lentivirus expressing either Ubqln2 -targeting shRNA (shUbqln2) or a control shRNA (shCtrl). Five days after transduction cells were fixed and immunostained for Ubqln2 and Map1b. Maximum intensity projections of z-stack images. Scale bar: 20 μm. (F) Primary cortical rat neurons were transduced with lentivirus co-expressing either Ubqln2 -targeting shRNA (shUbqln2) or a control shRNA (shCtrl) and tagRFP. Five days after transduction cells were harvested for immunoblotting. (F, G) Quantification of Ubqln2 and Map1b protein levels from (F). Data represent mean ± SD. Statistical analysis (n = 3) of the target protein/control protein ratio was performed using t test. (H) Representative images of UBQLN2 WT and KO HeLa cells fixed and immunostained for acetylated tubulin (Ac-Tubulin) and α-tubulin. Maximum intensity projections of z-stack images. Scale bar: 20 μm. (I) Quantification of Ac-Tubulin, α-tubulin, and Ac-Tubulin/α-tubulin ratio. Data represent mean gray intensity levels ± SD. Statistical analysis (n = 5, 20 cells each) was performed the using t test.

Article Snippet: Rabbit anti-MAP1B , Sigma-Aldrich , HPA022275.

Techniques: Western Blot, Transfection, Transduction, Expressing, shRNA

Journal: Life Science Alliance

Article Title: Multi-omics profiling identifies a deregulated FUS-MAP1B axis in ALS/FTD–associated UBQLN2 mutants

doi: 10.26508/lsa.202101327

Figure Lengend Snippet: (A) Ubqln2 knockdown in primary hippocampal mouse neurons after transduction with either Ubqln2 -targeting shRNA (shUbqln2) or a control shRNA (shCtrl). Cells were fixed and immunostained for Ubqln2. Transfected cells show an RFP signal. Scale bar: 20 μm. (B) Quantification of mean fluorescence intensity normalized to RFP fluorescence revealed a significant reduction of Ubqln2 abundance in shUbqln2 neurons compared with shCtrl in primary hippocampal neurons. Data represent mean ± SD ( t test, n = 28 cells per genotype). (C) Quantitative PCR of Map1b normalized to Gapdh showed a sevenfold enrichment of Map1b RNA in Ubqln2 knockdown primary cortical neurons compared with shCtrl.

Article Snippet: Rabbit anti-MAP1B , Sigma-Aldrich , HPA022275.

Techniques: Transduction, shRNA, Transfection, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Life Science Alliance

Article Title: Multi-omics profiling identifies a deregulated FUS-MAP1B axis in ALS/FTD–associated UBQLN2 mutants

doi: 10.26508/lsa.202101327

Figure Lengend Snippet: (A) Immunoblot of HeLa cells stably expressing HA-MAP1B. (B) Immunoprecipitation (IP)–mass spectrometry (MS) workflow in empty and stably HA-MAP1B-expressing HeLa cells. (C) MAP1B interactome. Lysates derived from empty and HA-MAP1B–expressing HeLa cells (n = 4) were subjected to HA-IP followed by MS and label-free quantification. MAP1B interaction candidates are shown in the upper right quadrant of the scatterplot (log 2 fold change (FC) ≥ 1; P ≤ 0.05, t test). Highlighted are components of the ubiquitin–proteasome system, chaperone, and cytoskeleton.

Article Snippet: Rabbit anti-MAP1B , Sigma-Aldrich , HPA022275.

Techniques: Western Blot, Stable Transfection, Expressing, Immunoprecipitation, Mass Spectrometry, Derivative Assay

Journal: Life Science Alliance

Article Title: Multi-omics profiling identifies a deregulated FUS-MAP1B axis in ALS/FTD–associated UBQLN2 mutants

doi: 10.26508/lsa.202101327

Figure Lengend Snippet: (A) SILAC-based phosphoproteomics workflow in UBQLN2 WT and ALS mutant LCLs (n = 4). (B) Combined numbers of identified and quantified phosphosites. Data-filtering steps are indicated. (C) Distribution of pSer, pThr, and pTyr sites in T487I and P497S UBQLN2 ALS mutants. (D) Changes in cellular phosphorylation in patient-derived UBQLN2-mutant LCLs. Median log 2 fold ratios in phosphorylation of the identified phosphosites are presented in relation to the intensity of detection. Hypophosphorylated peptides are shown in blue and hyper-phosphorylated ones in rede. Significantly changed MAP1B phosphosites are highlighted. (E) Gene Ontology (GO) enrichment analysis of proteins whose phosphorylation status was altered significantly in both mutants in the same direction with at least one mutant exceeding a log 2 fold change of ≥ 1 or ≤ −1. BP, biological process; CC, cellular component; MF, molecular function. (F) Protein–protein interaction (PPI) network of proteins whose phosphorylation status was significantly changed in both mutants with at least one mutant exceeding a log 2 ratio of ≥ 1 or ≤ −1. Significantly up- and down-regulated phosphosites are marked in red and blue, respectively.

Article Snippet: Rabbit anti-MAP1B , Sigma-Aldrich , HPA022275.

Techniques: Mutagenesis, Derivative Assay

Journal: Life Science Alliance

Article Title: Multi-omics profiling identifies a deregulated FUS-MAP1B axis in ALS/FTD–associated UBQLN2 mutants

doi: 10.26508/lsa.202101327

Figure Lengend Snippet: (A) Schematic representation of FUS. SYGQ-rich, serine, tyrosine, glycine, glutamine-rich domain; RRM, RNA recognition motif; RGG, arginine-glycine-glycine–rich region; NLS, nuclear localization signal; ZnF, zinc finger domain. (B) Immunoblot analysis of the Phos-tag gel– (upper panel) and SDS–PAGE (lower panel)–separated lysates derived from UBQLN2 WT and amyotrophic lateral sclerosis–mutant LCLs. (C) Immunoblot of UBQLN2 KO HeLa cells treated with two different siRNAs targeting FUS or a nontargeting siRNA control (siCtrl). (C, D) Quantification of MAP1B and FUS protein levels from (C). Data represent mean ± SD. Statistical analysis (n = 3) of the target protein/control protein ratio was performed using one-way ANOVA followed by Dunnett’s post hoc test. (E) SDS–PAGE of recombinant FUS variants (WT, S439A, S439E) stained with Coomassie blue. (F) Electrophoretic mobility shift assay (EMSA) of MBP-FUS-His 6 variants (WT, S439A, S439E) and SON pre-mRNA containing the stem loop and a downstream GUU (5 nM) (n = 3). (G) Immunoblot of FUS WT and KO HeLa cells. (H) FUS WT and KO cells transfected with HA-FUS proteoforms (WT and S439A) or left untreated (MOCK; only Lipofectamine). (I) Quantification of MAP1B protein levels upon expression of HA-FUS WT and S439A from (I). Data represent mean ± SD. Statistical analysis (n = 3) of the target protein/control protein ratio was performed using t test. (J) Working model: in cells with UBQLN2 WT, FUS S439 is constitutively phosphorylated, a state where FUS-RNA binding is impaired. UBQLN2 mutations (P497S and T497I) result in a reduction of pS439 and in elevated MAP1B levels, which are also observed upon UBQLN2 KO. Depending on whether UBQLN2 is functional or defective, KO of FUS has opposing effects on MAP1B levels.

Article Snippet: Rabbit anti-MAP1B , Sigma-Aldrich , HPA022275.

Techniques: Western Blot, SDS Page, Derivative Assay, Mutagenesis, Recombinant, Staining, Electrophoretic Mobility Shift Assay, Transfection, Expressing, RNA Binding Assay, Functional Assay

Journal: Life Science Alliance

Article Title: Multi-omics profiling identifies a deregulated FUS-MAP1B axis in ALS/FTD–associated UBQLN2 mutants

doi: 10.26508/lsa.202101327

Figure Lengend Snippet: Identified binding sites for wild-type FUS (blue) and cytoplasmically mislocalized mutant FUS (green) within the MAP1B gene from a published FUS PAR-CLIP data set . FUS-binding sites were defined as clusters of 10 or more overlapping reads of which at least 25% contained C-to-T mutations. Published BED files containing identified binding sites were uploaded as custom tracks to the UCSC genome browser, assembly NCBI36/hg18.

Article Snippet: Rabbit anti-MAP1B , Sigma-Aldrich , HPA022275.

Techniques: Binding Assay, Mutagenesis

Journal: Life Science Alliance

Article Title: Multi-omics profiling identifies a deregulated FUS-MAP1B axis in ALS/FTD–associated UBQLN2 mutants

doi: 10.26508/lsa.202101327

Figure Lengend Snippet: Table of Reagents.

Article Snippet: Rabbit anti-MAP1B , Sigma-Aldrich , HPA022275.

Techniques: Recombinant, Sequencing, Plasmid Preparation, Mutagenesis, shRNA, Transfection, Protease Inhibitor, Software, Picogreen Assay, DNA Extraction, Purification, Isolation, Mass Spectrometry

Journal: iScience

Article Title: Single-cell atlas unveils cellular heterogeneity and novel markers in human neonatal and adult intervertebral discs

doi: 10.1016/j.isci.2022.104504

Figure Lengend Snippet: Identification of NC Subpopulations and Their Pathway Network Analysis (A) The two NC subpopulations in UMAP and their expression of classical markers in both neonatal and adult IVD; two MAP1B +/SOX4+ NC subpopulations in the UMAP are colored green, and all other cells are colored gray; violin plots show NC expression levels of classical markers of NCs and immune cells for human neonatal and adult IVD. Other cells refer to the expression level for all cells in all clusters other than NC1 and NC2. (B) The immunostaining of classical NC and immune markers in neonatal and adult IVD are shown. Scale bar = 100 μm; original magnification = 20x. (C) Volcano plots showing the enriched genes for the entire populations of NC, subpopulations of NC1 or NC2, in neonatal or adult IVD. The y axis in the volcano plots shows the -log 10 p where the p value was about an enriched gene expression level in a specific subpopulation in a specific age (neonatal or adult) against all other cells the same age. The x-axis shows the log 2 FC (FC - fold change) of the expression level. The cut-off threshold was set to FC > 2 and p < 1×10 −24 . The enriched genes meeting the cut-off threshold are colored with red. The genes not meeting the cut-off threshold are colored with gray.

Article Snippet: Rabbit Polyclonal Anti-MAP1B , Novus Biologicals , NBP3-04801-20ul.

Techniques: Expressing, Immunostaining

Journal: iScience

Article Title: Single-cell atlas unveils cellular heterogeneity and novel markers in human neonatal and adult intervertebral discs

doi: 10.1016/j.isci.2022.104504

Figure Lengend Snippet: Heterogeneity of Annulus Fibrosus Cells in Human Neonatal and Adult IVDs (A and B) Expression levels of classical AFC markers ( COL1A1 , CALR , and HSPA6 ), classical NPC markers (ACAN , COL2A1 , and SOX9 ), and classical NC markers ( MAP1B ) are shown for comparison, for both (A) neonatal IVD and (B) adult IVD. (C) The AFC populations showed strong heterogeneity in neonatal IVD by having 4 distinct subtypes and their heterogenous markers. We demonstrated the expression distribution projected on UMAP and their quantitative expression levels. (D) Among the 5 heterogeneous subpopulations detected in (C), the neonatal AFCs exhibited a decreasing trend of expression levels of ECM-relevant, collagen-producing genes from inner core NPCs to the outer region AFCs following the order of iAFC, oAFC1-3. (E) The biological function and key canonical pathway enrichment results followed the same gradient. (F) Predicted scheme for spatial distribution of the 4 AFC populations, along with inner NPC core and notochord, in neonatal IVD, as compared with the classical IVD structure in adult humans.

Article Snippet: Rabbit Polyclonal Anti-MAP1B , Novus Biologicals , NBP3-04801-20ul.

Techniques: Expressing, Comparison

Journal: iScience

Article Title: Single-cell atlas unveils cellular heterogeneity and novel markers in human neonatal and adult intervertebral discs

doi: 10.1016/j.isci.2022.104504

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal Anti-MAP1B , Novus Biologicals , NBP3-04801-20ul.

Techniques: Recombinant, Expressing, Software