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anln  (Proteintech)
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Proteintech anln
( A ) Immunoblotting of myelin purified from the brains of wild type mice at P75 compared to brain lysates indicates that anillin <t>(ANLN)</t> is enriched in myelin similar to septin 8 variant 1 (SEPT8_v1). The same amount of protein was loaded. The myelin marker MOG and the axonal marker TUJ1 served as controls. Blot shows n = 2 mice per genotype representative of n = 3 mice per genotype. ( B–C ) Immunofluorescent signal of ANLN (green in B ) <t>and</t> <t>SEPT7</t> (green in C ) extends longitudinally along axons identified by neurofilament-labelling (red in B–C ). Additional ANLN-immunopositive puncta (asterisks in B ) were not evidently associated with filamentous structures (arrowheads in B,C ). The panels show maximal projections of confocal stacks and 3-dimensional reconstructions of longitudinally sectioned spinal cord of P75 WT mice. Images representative of three mice. ( D ) Immunoblotting of myelin purified from the brains of wild-type mice at P15, P18, P21 and P24 indicates that the abundance of ANLN in myelin increases with maturation. Myelin septins (SEPT2, SEPT4, SEPT7, SEPT8) and MAG served as control. Blot shows n = 1 mouse per timepoint. ( E ) Immunolabelling of longitudinally sectioned WT optic nerves detects ANLN (red) in proximity to SEPT8 (green); co-labeled structures (arrowheads) were seen occasionally at P21 and frequently at P28 but not at P15. TUJ1 served as axonal marker. Images representative of three experiments.
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( A ) Immunoblotting of myelin purified from the brains of wild type mice at P75 compared to brain lysates indicates that anillin (ANLN) is enriched in myelin similar to septin 8 variant 1 (SEPT8_v1). The same amount of protein was loaded. The myelin marker MOG and the axonal marker TUJ1 served as controls. Blot shows n = 2 mice per genotype representative of n = 3 mice per genotype. ( B–C ) Immunofluorescent signal of ANLN (green in B ) and SEPT7 (green in C ) extends longitudinally along axons identified by neurofilament-labelling (red in B–C ). Additional ANLN-immunopositive puncta (asterisks in B ) were not evidently associated with filamentous structures (arrowheads in B,C ). The panels show maximal projections of confocal stacks and 3-dimensional reconstructions of longitudinally sectioned spinal cord of P75 WT mice. Images representative of three mice. ( D ) Immunoblotting of myelin purified from the brains of wild-type mice at P15, P18, P21 and P24 indicates that the abundance of ANLN in myelin increases with maturation. Myelin septins (SEPT2, SEPT4, SEPT7, SEPT8) and MAG served as control. Blot shows n = 1 mouse per timepoint. ( E ) Immunolabelling of longitudinally sectioned WT optic nerves detects ANLN (red) in proximity to SEPT8 (green); co-labeled structures (arrowheads) were seen occasionally at P21 and frequently at P28 but not at P15. TUJ1 served as axonal marker. Images representative of three experiments.

Journal: eLife

Article Title: Anillin facilitates septin assembly to prevent pathological outfoldings of central nervous system myelin

doi: 10.7554/eLife.43888

Figure Lengend Snippet: ( A ) Immunoblotting of myelin purified from the brains of wild type mice at P75 compared to brain lysates indicates that anillin (ANLN) is enriched in myelin similar to septin 8 variant 1 (SEPT8_v1). The same amount of protein was loaded. The myelin marker MOG and the axonal marker TUJ1 served as controls. Blot shows n = 2 mice per genotype representative of n = 3 mice per genotype. ( B–C ) Immunofluorescent signal of ANLN (green in B ) and SEPT7 (green in C ) extends longitudinally along axons identified by neurofilament-labelling (red in B–C ). Additional ANLN-immunopositive puncta (asterisks in B ) were not evidently associated with filamentous structures (arrowheads in B,C ). The panels show maximal projections of confocal stacks and 3-dimensional reconstructions of longitudinally sectioned spinal cord of P75 WT mice. Images representative of three mice. ( D ) Immunoblotting of myelin purified from the brains of wild-type mice at P15, P18, P21 and P24 indicates that the abundance of ANLN in myelin increases with maturation. Myelin septins (SEPT2, SEPT4, SEPT7, SEPT8) and MAG served as control. Blot shows n = 1 mouse per timepoint. ( E ) Immunolabelling of longitudinally sectioned WT optic nerves detects ANLN (red) in proximity to SEPT8 (green); co-labeled structures (arrowheads) were seen occasionally at P21 and frequently at P28 but not at P15. TUJ1 served as axonal marker. Images representative of three experiments.

Article Snippet: Antibodies were specific for ANLN (Acris AP16165PU-N; 1:200), SEPT7 (IBL18991; 1:1000), SEPT8 (ProteinTech Group 11769–1-AP; 1:500), TUJ1 (Covance MMS-435P; 1:1000), neurofilament (Covance SMI-31; 1:1500), myelin-associated glycoprotein (MAG clone 513; Chemicon MAB1567; 1:50), voltage-gated sodium channel Na v 1,6 (alomonelabs ASC-009; 1:500) or contactin-associated protein (CASPR; Neuromabs 75–001; 1:500).

Techniques: Western Blot, Purification, Variant Assay, Marker, Control, Labeling

( A–B ) Immunolabelling of coronal brain sections of WT mice at P75 detects ANLN (red) and SEPT8 (green) filaments (arrowheads) in the indicated white matter tracts. ( A–A’ ) shows labelling in the fimbria. ( A’ ), enlargement of the dashed square in ( A ). ( B ) shows labelling in the corpus callosum. MAG or neurofilaments (NF) were co-labelled as control. Images representative of three experiments.

Journal: eLife

Article Title: Anillin facilitates septin assembly to prevent pathological outfoldings of central nervous system myelin

doi: 10.7554/eLife.43888

Figure Lengend Snippet: ( A–B ) Immunolabelling of coronal brain sections of WT mice at P75 detects ANLN (red) and SEPT8 (green) filaments (arrowheads) in the indicated white matter tracts. ( A–A’ ) shows labelling in the fimbria. ( A’ ), enlargement of the dashed square in ( A ). ( B ) shows labelling in the corpus callosum. MAG or neurofilaments (NF) were co-labelled as control. Images representative of three experiments.

Article Snippet: Antibodies were specific for ANLN (Acris AP16165PU-N; 1:200), SEPT7 (IBL18991; 1:1000), SEPT8 (ProteinTech Group 11769–1-AP; 1:500), TUJ1 (Covance MMS-435P; 1:1000), neurofilament (Covance SMI-31; 1:1500), myelin-associated glycoprotein (MAG clone 513; Chemicon MAB1567; 1:50), voltage-gated sodium channel Na v 1,6 (alomonelabs ASC-009; 1:500) or contactin-associated protein (CASPR; Neuromabs 75–001; 1:500).

Techniques: Control

( A ) Scheme of the wild type (WT) Anln gene and the engineered Anln allele before and after recombination. Exon 4 of the Anln flox -allele is flanked by loxP-sites (floxed) for Cre-mediated recombination. Positions of PCR primers ( P1, P2, P3 ) are indicated. ( B ) Genotyping PCR of DNA isolated from mouse tail tips identifies the indicated alleles. Gel shows n = 2 mice per genotype. ( C ) Immunoblot analysis of brain lysates and myelin purified from P75 control ( Anln fl/fl ) and Anln fl/fl ;Cnp Cre/WT ( Anln cKO) mice. Note that ANLN is only detectable in myelin purified from the brains of control mice. Note that the abundance of SEPT8 is reduced in myelin purified from Anln cKO compared to control mice. MOG and TUJ1 were detected as controls. Blot shows n = 2 mice per genotype. ( D ) Electron micrographs of optic nerves at eight mo indicates unaltered myelin periodicity and compaction in Anln cKO mice. Representative of three mice per genotype.

Journal: eLife

Article Title: Anillin facilitates septin assembly to prevent pathological outfoldings of central nervous system myelin

doi: 10.7554/eLife.43888

Figure Lengend Snippet: ( A ) Scheme of the wild type (WT) Anln gene and the engineered Anln allele before and after recombination. Exon 4 of the Anln flox -allele is flanked by loxP-sites (floxed) for Cre-mediated recombination. Positions of PCR primers ( P1, P2, P3 ) are indicated. ( B ) Genotyping PCR of DNA isolated from mouse tail tips identifies the indicated alleles. Gel shows n = 2 mice per genotype. ( C ) Immunoblot analysis of brain lysates and myelin purified from P75 control ( Anln fl/fl ) and Anln fl/fl ;Cnp Cre/WT ( Anln cKO) mice. Note that ANLN is only detectable in myelin purified from the brains of control mice. Note that the abundance of SEPT8 is reduced in myelin purified from Anln cKO compared to control mice. MOG and TUJ1 were detected as controls. Blot shows n = 2 mice per genotype. ( D ) Electron micrographs of optic nerves at eight mo indicates unaltered myelin periodicity and compaction in Anln cKO mice. Representative of three mice per genotype.

Article Snippet: Antibodies were specific for ANLN (Acris AP16165PU-N; 1:200), SEPT7 (IBL18991; 1:1000), SEPT8 (ProteinTech Group 11769–1-AP; 1:500), TUJ1 (Covance MMS-435P; 1:1000), neurofilament (Covance SMI-31; 1:1500), myelin-associated glycoprotein (MAG clone 513; Chemicon MAB1567; 1:50), voltage-gated sodium channel Na v 1,6 (alomonelabs ASC-009; 1:500) or contactin-associated protein (CASPR; Neuromabs 75–001; 1:500).

Techniques: Isolation, Western Blot, Purification, Control

( A ) Differential myelin proteome analysis indicates that ANLN is readily identified in myelin purified from the brains of control mice at P75 but undetectable in Anln fl/fl ;Cnp Cre/WT ( Anln cKO)-myelin. Mean +/SEM; n = 3 mice per genotype. ( B ) Differential myelin proteome analysis reveals that the abundance of myelin septins (SEPT2, SEPT4, SEPT7, SEPT8) is diminished in myelin purified from Anln cKO-mice at P75 compared to Anln fl/fl mice. The abundance of the putative septin-associated proteins RHOB and CDC42 is also significantly reduced in Anln cKO-myelin, although to a lesser degree. The horizontal black dashed line marks a threshold indicating halved abundance of a protein in myelin. Mean +/SEM; n = 3 mice per genotype; For q-values as calculated by R data analysis (see Materials and Methods for details) see . ( C ) Differential myelin proteome analysis indicates that the abundance of classical myelin proteins is unaltered in Anln cKO-myelin. Mean +/SEM. n = 3 mice per genotype; not significant according to q-value calculation using R (see Materials and Methods for details); for exact q-values see . ( D ) By differential myelin proteome analysis, the abundance of cytoskeletal proteins is unaltered in Anln cKO-myelin, except for myelin septins and septin-associated proteins as highlighted in ( B ). Mean +/SEM. n = 3 mice per genotype; not significant according to q-value calculation using R; for exact q-values see . ( E ) qRT-PCR to determine the abundance of mRNAs encoding putative septin-associated proteins RHOB and CDC42 in the white matter (corpus callosum) of control ( Anln fl/fl ) versus Anln cKO-mice. Note that Rhob and Cdc42 mRNAs were unaltered in abundance. Mean +/SEM. n = 6 mice per genotype; two-way ANOVA; RhoB p>0.9999, Cdc42 p>0.9999.

Journal: eLife

Article Title: Anillin facilitates septin assembly to prevent pathological outfoldings of central nervous system myelin

doi: 10.7554/eLife.43888

Figure Lengend Snippet: ( A ) Differential myelin proteome analysis indicates that ANLN is readily identified in myelin purified from the brains of control mice at P75 but undetectable in Anln fl/fl ;Cnp Cre/WT ( Anln cKO)-myelin. Mean +/SEM; n = 3 mice per genotype. ( B ) Differential myelin proteome analysis reveals that the abundance of myelin septins (SEPT2, SEPT4, SEPT7, SEPT8) is diminished in myelin purified from Anln cKO-mice at P75 compared to Anln fl/fl mice. The abundance of the putative septin-associated proteins RHOB and CDC42 is also significantly reduced in Anln cKO-myelin, although to a lesser degree. The horizontal black dashed line marks a threshold indicating halved abundance of a protein in myelin. Mean +/SEM; n = 3 mice per genotype; For q-values as calculated by R data analysis (see Materials and Methods for details) see . ( C ) Differential myelin proteome analysis indicates that the abundance of classical myelin proteins is unaltered in Anln cKO-myelin. Mean +/SEM. n = 3 mice per genotype; not significant according to q-value calculation using R (see Materials and Methods for details); for exact q-values see . ( D ) By differential myelin proteome analysis, the abundance of cytoskeletal proteins is unaltered in Anln cKO-myelin, except for myelin septins and septin-associated proteins as highlighted in ( B ). Mean +/SEM. n = 3 mice per genotype; not significant according to q-value calculation using R; for exact q-values see . ( E ) qRT-PCR to determine the abundance of mRNAs encoding putative septin-associated proteins RHOB and CDC42 in the white matter (corpus callosum) of control ( Anln fl/fl ) versus Anln cKO-mice. Note that Rhob and Cdc42 mRNAs were unaltered in abundance. Mean +/SEM. n = 6 mice per genotype; two-way ANOVA; RhoB p>0.9999, Cdc42 p>0.9999.

Article Snippet: Antibodies were specific for ANLN (Acris AP16165PU-N; 1:200), SEPT7 (IBL18991; 1:1000), SEPT8 (ProteinTech Group 11769–1-AP; 1:500), TUJ1 (Covance MMS-435P; 1:1000), neurofilament (Covance SMI-31; 1:1500), myelin-associated glycoprotein (MAG clone 513; Chemicon MAB1567; 1:50), voltage-gated sodium channel Na v 1,6 (alomonelabs ASC-009; 1:500) or contactin-associated protein (CASPR; Neuromabs 75–001; 1:500).

Techniques: Purification, Control, Quantitative RT-PCR

( A ) Volcano plot summarizing genotype-dependent quantitative myelin proteome analysis. Data points represent quantified proteins in myelin purified at P75 from the brains of Anln cKO compared to Anln fl/fl mice (n = 3 mice per genotype). Data points are plotted as log2-transformed fold-change (FC) on the x-axis against the −log10-transformed q-value on the y-axis. The horizontal red dashed line indicates a q-value of q = 0.01; the vertical black dashed lines mark the ±1 log2 fold-change threshold indicating a halved or doubled abundance of a protein in myelin, respectively. Data points representing myelin septin monomers (SEPT2, SEPT4, SEPT7, SEPT8) are highlighted in light red color with protein names given; note that their abundance is strongly reduced in Anln cKO compared to Anln fl/fl myelin. Also note that ANLN is not represented because it was not detected in Anln cKO myelin. For bar graphs showing genotype-dependent comparison of the abundance of individual proteins in myelin see . For the original dataset and exact q-values see . ( B ) Immunoblotting validates the lack of anillin (ANLN) and the strong reduction of septins (SEPT2, SEPT4, SEPT7, SEPT8) in myelin purified from the brains of Anln cKO-mice. ATPase Na+/K + transporting subunit alpha 3 (ATP1A3) was detected as control. Blot shows n = 3 mice per genotype. ( C ) Immunoblotting indicates that the abundance of classical myelin proteins (PLP/DM20, SIRT2, CD9, CA2) is unaltered in myelin purified from the brains of Anln cKO-mice. ATP1A1 served as control. Blot shows n = 3 mice per genotype. ( D ) Genotype-dependent quantitative assessment of PtdIns(4,5)P 2 (PIP 2 )–levels in myelin purified from the brains of Anln cKO-mice compared to controls ( Anln fl/fl ) at P75. Mean +/SEM. n = 6 mice per genotype; two-tailed unpaired t-test; PtdIns(4,5)P 2 p=0.0435. ( E ) qRT-PCR to determine the abundance of mRNAs encoding anillin and myelin septins in the white matter (corpus callosum) of control ( Anln fl/fl ) versus Anln cKO-mice. Note that Anln mRNA was virtually undetectable in Anln cKO-mice while the abundances of Sept2 , Sept4 , Sept7 and Sept8 mRNAs were unaltered. Mean +/SEM. n = 6 mice per genotype; two-way ANOVA; Anln p<0.0001, Sept2 p>0.9999, Sept4 p>0.9999, Sept7 p>0.9999, Sept8 p>0.9999. 10.7554/eLife.43888.010 Figure 3—source data 1. Label-free quantification of proteins in myelin purified from the brains of Anln cKO and control mice Tryptic peptides derived from two technical replicates (replicate digestion) per biological replicate (n = 3 mice per genotype) were analyzed by LC-MS (12 runs in total). Proteins (FDR < 1%; two peptides/protein) and peptides (FDR < 1%;≥6 amino acids) were identified by database search against the UniprotKB/SwissProt mouse database using PLGS. Data were post-processed with the software package ISOQuant to calculate absolute in-sample amounts for each detected protein based on the TOP3 approach. Reported abundance values are defined as the relative amount of each protein in respect to the sum over all detected proteins (ppm: parts per million (w/w) of total protein). The typical contaminant proteins trypsin and keratins were filtered. One technical replicate of a control sample was identified as outlier based on its low correlation coefficient of ≤0.76 (all other runs ≥ 0.95) and thus excluded from analysis.

Journal: eLife

Article Title: Anillin facilitates septin assembly to prevent pathological outfoldings of central nervous system myelin

doi: 10.7554/eLife.43888

Figure Lengend Snippet: ( A ) Volcano plot summarizing genotype-dependent quantitative myelin proteome analysis. Data points represent quantified proteins in myelin purified at P75 from the brains of Anln cKO compared to Anln fl/fl mice (n = 3 mice per genotype). Data points are plotted as log2-transformed fold-change (FC) on the x-axis against the −log10-transformed q-value on the y-axis. The horizontal red dashed line indicates a q-value of q = 0.01; the vertical black dashed lines mark the ±1 log2 fold-change threshold indicating a halved or doubled abundance of a protein in myelin, respectively. Data points representing myelin septin monomers (SEPT2, SEPT4, SEPT7, SEPT8) are highlighted in light red color with protein names given; note that their abundance is strongly reduced in Anln cKO compared to Anln fl/fl myelin. Also note that ANLN is not represented because it was not detected in Anln cKO myelin. For bar graphs showing genotype-dependent comparison of the abundance of individual proteins in myelin see . For the original dataset and exact q-values see . ( B ) Immunoblotting validates the lack of anillin (ANLN) and the strong reduction of septins (SEPT2, SEPT4, SEPT7, SEPT8) in myelin purified from the brains of Anln cKO-mice. ATPase Na+/K + transporting subunit alpha 3 (ATP1A3) was detected as control. Blot shows n = 3 mice per genotype. ( C ) Immunoblotting indicates that the abundance of classical myelin proteins (PLP/DM20, SIRT2, CD9, CA2) is unaltered in myelin purified from the brains of Anln cKO-mice. ATP1A1 served as control. Blot shows n = 3 mice per genotype. ( D ) Genotype-dependent quantitative assessment of PtdIns(4,5)P 2 (PIP 2 )–levels in myelin purified from the brains of Anln cKO-mice compared to controls ( Anln fl/fl ) at P75. Mean +/SEM. n = 6 mice per genotype; two-tailed unpaired t-test; PtdIns(4,5)P 2 p=0.0435. ( E ) qRT-PCR to determine the abundance of mRNAs encoding anillin and myelin septins in the white matter (corpus callosum) of control ( Anln fl/fl ) versus Anln cKO-mice. Note that Anln mRNA was virtually undetectable in Anln cKO-mice while the abundances of Sept2 , Sept4 , Sept7 and Sept8 mRNAs were unaltered. Mean +/SEM. n = 6 mice per genotype; two-way ANOVA; Anln p<0.0001, Sept2 p>0.9999, Sept4 p>0.9999, Sept7 p>0.9999, Sept8 p>0.9999. 10.7554/eLife.43888.010 Figure 3—source data 1. Label-free quantification of proteins in myelin purified from the brains of Anln cKO and control mice Tryptic peptides derived from two technical replicates (replicate digestion) per biological replicate (n = 3 mice per genotype) were analyzed by LC-MS (12 runs in total). Proteins (FDR < 1%; two peptides/protein) and peptides (FDR < 1%;≥6 amino acids) were identified by database search against the UniprotKB/SwissProt mouse database using PLGS. Data were post-processed with the software package ISOQuant to calculate absolute in-sample amounts for each detected protein based on the TOP3 approach. Reported abundance values are defined as the relative amount of each protein in respect to the sum over all detected proteins (ppm: parts per million (w/w) of total protein). The typical contaminant proteins trypsin and keratins were filtered. One technical replicate of a control sample was identified as outlier based on its low correlation coefficient of ≤0.76 (all other runs ≥ 0.95) and thus excluded from analysis.

Article Snippet: Antibodies were specific for ANLN (Acris AP16165PU-N; 1:200), SEPT7 (IBL18991; 1:1000), SEPT8 (ProteinTech Group 11769–1-AP; 1:500), TUJ1 (Covance MMS-435P; 1:1000), neurofilament (Covance SMI-31; 1:1500), myelin-associated glycoprotein (MAG clone 513; Chemicon MAB1567; 1:50), voltage-gated sodium channel Na v 1,6 (alomonelabs ASC-009; 1:500) or contactin-associated protein (CASPR; Neuromabs 75–001; 1:500).

Techniques: Purification, Transformation Assay, Comparison, Western Blot, Control, Two Tailed Test, Quantitative RT-PCR, Quantitative Proteomics, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Software