Journal: Endocrinology

Article Title: Deficiency of C1QL1 reduced murine ovarian follicle reserve through intraovarian and endocrine control.

doi: 10.1210/endocr/bqac048

Figure Lengend Snippet: Figure 5. Deficiency of C1QL1 promoted apoptosis and inhibited autophagy in the ovary. (A) Representative images of TUNEL staining of ovarian sections. The green signals indicate representative cells with positive TUNEL staining, which was mainly detected in the large atretic follicles. (B) The intensity of TUNEL-positive signals was shown in charts (n = 4 per group). (C) Representative images of immunofluorescence staining of ovarian sections with LC3B antibody. LC3B-positive signals were found in granulosa cells and oocytes and were weakened in C1QL1-deficient ovaries. (D) The intensity of LC3B-positive signals are shown (n = 4 per group). (E) Apoptosis- and autophagy-related proteins in the ovaries were analyzed by Western blotting. Representative blots are shown in the left panel, and the amount of protein normalized to β-tubulin, which is presented as mean ± SEM, is shown in the right panel. n = 6 per group. *P < 0.05, **P < 0.01, ***P < 0.001. NS, no significance.

Article Snippet: The sections and granulosa cells were stained with anti-rabbit LC3B antibody and Alexa Fluor 594-conjugated secondary antibody and mounted with DAPI (Boster).

Techniques: TUNEL Assay, Staining, Immunofluorescence, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: SerThr-PhosphoProteome of Brain from Aged PINK1-KO+A53T-SNCA Mice Reveals pT1928-MAP1B and pS3781-ANK2 Deficits, as Hub between Autophagy and Synapse Changes

doi: 10.3390/ijms20133284

Figure Lengend Snippet: Phosphoproteomics revealed a cluster of dysregulations (downregulations in blue, upregulations in red color) in factors associated with microtubules and microfilaments. The list also presents signal intensity and coefficient of variance (CV), the phosphorylation site, the peptide sequence around the phosphorylated residue (illustrated by asterisk *), and the peptide count. A selective massive deficit of pT1928 in microtubule-associated protein 1B (MAP1B) and the second strongest deficit of pS3781 in ANK2 as a MAP1B-interactor were documented. These prominent changes clustered with an enrichment of factors that link the cytoskeleton to either the mitochondria or to the plasma membrane signaling apparatus, namely MAP2, EPB41L2 (4.1G), MARK1, MAP1A, KIF1A, RP1, ADD2, CLASP1, and EPB41L1 (4.1N), see Figure 3A.

Article Snippet: Quantitative immunoblots of MAP1B were performed as in the previous description of the DM mice [ ], employing the anti-MAP1B antibody from BD Biosciences (San Jose, CA, USA, catalog # 612678), the anti-ACTB antibody from Sigma (Ronkonkoma, NY, USA, # A5441), and the anti-TUBA antibody from Abcam (Cambridge, UK, # ab15246) at the recommended dilutions.

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: SerThr-PhosphoProteome of Brain from Aged PINK1-KO+A53T-SNCA Mice Reveals pT1928-MAP1B and pS3781-ANK2 Deficits, as Hub between Autophagy and Synapse Changes

doi: 10.3390/ijms20133284

Figure Lengend Snippet: ( A ) Scheme of MAP1B protein sequence, cleavage into heavy chain and light chain 1 (LC1), structural motifs with their interactions (LC1 binding in heavy chain, actin binding domain ABD, microtubule binding domain MBD, microtubule assembly helping site MTA, region that contains 12 repeats of a 17mer sequence, MAP1A interacting domain), and characterized GSK3beta-dependent phospho-sites at pS1260/pT1265, which have an established effect on cytoskeletal reorganization. Alignment of MAP1B protein sequences across species illustrates the conservation of the DM-modulated Thr/Ser site (red arrow) within the 17mer repeat (highlighted as black bars above sequence) of unknown function. The symbols * : . below the amino acid alignment highlight different phylogenetic conservation. ( B ) Scheme of ANK2 protein sequence, structural motifs (Ankyrin repeat, ZU5 domains, and DEATH domain, with characterized phosphorylation sites at S846 and S3850. Alignment of ANK2 protein sequences across species illustrates the conservation of the DM-modulated Ser site (red arrow) within the C-terminal target interaction domain. For a complete list of the residues with potential phosphorylation, see www.phosphosite.org .

Article Snippet: Quantitative immunoblots of MAP1B were performed as in the previous description of the DM mice [ ], employing the anti-MAP1B antibody from BD Biosciences (San Jose, CA, USA, catalog # 612678), the anti-ACTB antibody from Sigma (Ronkonkoma, NY, USA, # A5441), and the anti-TUBA antibody from Abcam (Cambridge, UK, # ab15246) at the recommended dilutions.

Techniques: Sequencing, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: SerThr-PhosphoProteome of Brain from Aged PINK1-KO+A53T-SNCA Mice Reveals pT1928-MAP1B and pS3781-ANK2 Deficits, as Hub between Autophagy and Synapse Changes

doi: 10.3390/ijms20133284

Figure Lengend Snippet: ( A ) Schematic representation of dysregulated factors, based on their subcellular localization and on textmining about functional interaction effects. Three cytoskeletal layers underlying neuronal plasma membranes (microtubules, actin-microfilaments, spectrin meshwork) are shown, which provide correct localization to mitochondria, vesicles, and the membrane channels/pumps. The overexpressed mutant A53T-SNCA with its aggregates, the deleted PINK1 kinase, and the downstream proteins affected by altered phosphorylation—and LRRK2 as a DAPK2 homologous kinase that phosphorylates MAP1B in abnormal manner in the PARK8 variant of PD—are visualized as colored symbols, reminiscent of their known 3D structures and with rough representation of their size differences. The two strongest deficits in phosphorylation are highlighted in pink/yellow circles. ( B ) As additional automated scheme on protein–protein interactions and pathway enrichments, search tool for the retrieval of interacting genes (STRING) diagrams are provided for all 45 factors with reduced Ser/Thr-phosphorylation and ( C ) 49 factors with increased Ser/Thr-phosphorylation. The protein–protein enrichment p -value was very significant among the downregulations ( p = 7.54 × 10 −12 ), but barely significant among the downregulations ( p = 0.036). Previous knowledge of the interaction between these factors from experiments, co-expression, or text mining is illustrated by connecting lines of various colors.

Article Snippet: Quantitative immunoblots of MAP1B were performed as in the previous description of the DM mice [ ], employing the anti-MAP1B antibody from BD Biosciences (San Jose, CA, USA, catalog # 612678), the anti-ACTB antibody from Sigma (Ronkonkoma, NY, USA, # A5441), and the anti-TUBA antibody from Abcam (Cambridge, UK, # ab15246) at the recommended dilutions.

Techniques: Functional Assay, Mutagenesis, Variant Assay, Protein Enrichment, Expressing

Journal: International Journal of Molecular Sciences

Article Title: SerThr-PhosphoProteome of Brain from Aged PINK1-KO+A53T-SNCA Mice Reveals pT1928-MAP1B and pS3781-ANK2 Deficits, as Hub between Autophagy and Synapse Changes

doi: 10.3390/ijms20133284

Figure Lengend Snippet: Validation experiments regarding expression modulation at protein and mRNA level. ( A ) Quantitative immunoblots detecting MAP1B relative to beta-actin (ATCB) as loading control and marker of the cortical actin cytoskeleton, or to alpha-tubulin (TUBA) as loading control and marker of the microtubule cytoskeleton, both for RIPA-soluble and for SDS-soluble fractions of the brain proteome. ( B ) Quantitative immunoblots detecting ANK2 relative to beta-actin (ATCB) as loading control or to alpha-tubulin (TUBA) as loading control, again for RIPA- and SDS-soluble fractions. ( C ) Transcript abundance of PINK1 (above) and MAP1B (below), normalized to the loading control HPRT1 , in human SH-SY5Y neuroblastoma cells undergoing nutrient deprivation in HBSS medium without fetal calf serum over 48 h. Black bars represent stable knock-down (KD) of PINK1 , while white bars represent non-target (NT) knock-down control. Starvation induces PINK1 mRNA in phasic manner with a peak at 12–16 h. MAP1B mRNA is induced in a similar pattern, but not sustained after 12 h in PINK1-KD cells ( n = 4 PINK1 -KD versus 4 NT-KD). ( D ) Quantitative immunoblots in primary cortical neurons (28 days in vitro) starved for 2 h with HBSS medium show a significant reduction of the LC3-II isoform versus LC3-I in DM mice ( n = 4 DM versus 4 WT, two litters). Significance is illustrated by asterisks, with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Quantitative immunoblots of MAP1B were performed as in the previous description of the DM mice [ ], employing the anti-MAP1B antibody from BD Biosciences (San Jose, CA, USA, catalog # 612678), the anti-ACTB antibody from Sigma (Ronkonkoma, NY, USA, # A5441), and the anti-TUBA antibody from Abcam (Cambridge, UK, # ab15246) at the recommended dilutions.

Techniques: Expressing, Western Blot, Marker, In Vitro