Journal: bioRxiv

Article Title: E4F1 COORDINATES PYRUVATE METABOLISM AND THE ACTIVITY OF THE ELONGATOR COMPLEX TO ENSURE PROTEIN TRANSLATION FIDELITY DURING NEURONAL DEVELOPMENT

doi: 10.1101/2022.12.19.521032

Figure Lengend Snippet: (A) RT-qPCR analysis of E4f1 mRNA levels and those of genes encoding key subunits or regulators of the pyruvate dehydrogenase complex (PDC) ( Dlat, Dld, Mpc1, Pdpr , and Pdha1 ) in the forebrain of E14.5 E4f1 (Nes)KO and CTL embryos (n=5 animals/group). (B) Immunoblot analysis of E4F1, DLAT, MPC1, DLD, PDHA1, and ACTIN (loading control) protein levels in the brain of E14.5 E4f1 (Nes)KO and CTL embryos. Right panel: Histobars represent the quantification of immunoblots performed on n=4 independent samples/group. (C) PDH activity in protein extracts prepared from E18.5 E4f1 (Nes)KO and CTL embryos (n=5 animals/group). (D) Blood lactate levels in E18.5 E4f1 (Nes)KO and CTL embryos (n≥40 animals/group). (E) IF analysis of MCT4 protein levels in brain sagittal sections prepared from E14.5 E4f1 (Nes)KO and CTL embryos. Fb= forebrain; Mb= middle brain; LV= lateral ventricule. Scale bars, 50 μm. Data are presented as mean + standard error of mean (SEM) for and or mean + standard deviation (SD) for and from the indicated number of animals. Statistical significance was evaluated using unpaired bilateral Student’s t -test (ns, not significant).

Article Snippet: Total protein extracts were prepared from isolated brains lysed in Laemmli buffer (80mM Tris pH=6,8, 2% SDS, 12% sucrose, 2% β-mercaptoethanol, bromophenol blue) and immunoblotting was performed using the following antibodies: anti-E4F1 (1/1000) , C-CASP3 (Cell Signaling, 9661S, 1/1000), DLAT (Santa Cruz, sc-271534, 1/500), MPC1 (Sigma, HPA045119, 1/1000), DLD (Santa Cruz, sc-365977, 1/1000), PDHE1 (Life Technologies, 459400, 1/500), ELP3 (Abcam, ab190907, 1/500), ELP1 (Abcam, ab62498, 1/500), TUBULIN (Sigma, T6199, 1/1000) and ACTIN (Sigma, A3854, 1/7000).

Techniques: Quantitative RT-PCR, Western Blot, Control, Activity Assay, Standard Deviation

Journal: Biomolecules

Article Title: The Proteomic Landscape of Parkin-Deficient and Parkin-Overexpressing Rat Nucleus Accumbens: An Insight into the Role of Parkin in Methamphetamine Use Disorder

doi: 10.3390/biom15070958

Figure Lengend Snippet: Parkin Deficit or Overexpression in Rat Nucleus Accumbens Changes Aerobic Respiration in Opposite Directions. ( A , B ) Gene set enrichment analysis (GSEA)-enrichment plots showing protein sets from GOBP_AEROBIC RESPIRATION signature significantly overrepresented ( p < 0.05) in parkin knockout (PKO) or parkin overexpression (PO) rat nucleus accumbens, as compared to wild-type nucleus accumbens. ( C , D ) Hierarchical clustering heatmap of the leading-edge proteins from the GOBP_AEROBIC_RESPIRATION ( p < 0.05) in PKO NAc and PO NAc. Three Krebs cycle enzymes, citrate synthase (CS), mitochondrial malonate dehydrogenase (MDH2), and dihydrolipoamide S-succinyltransferase (DLST), were decreased in PKO NAc and increased in PO NAc relative to wild-type NAc (red arrows).

Article Snippet: Proteins were transferred to PVDF membrane (EMD Millipore, Burlington, MA, USA) at 400 mA, blocked with 5% non-fat dried milk dissolved in TBST (10 mM Tris, 150 mM NaCl, and 0.05% Tween-20), and incubated overnight at 4 °C with the following primary antibodies (diluted in 5% milk-containing TBST) against the following proteins: citrate synthase (CS) (NBP2-13878, 1:1000 Novus Biologicals, Centennial, CO, USA), dihydrolipoamide S-succinyltransferase (DLST) (PA5-51794, 1:1000,Invitrogen Life Technologies, Carlsbad, USA), mitochondrial malate dehydrogenase (MDH2) (9610, 1:1000, Cell Signaling, Danvers, MA, USA), dihydrolipoamide dehydrogenase (DLD) (NBP1-31302, 1:1000, Novus Biologicals, Centennial, CO, USA), 2-oxoglutarate dehydrogenase (OGDH) (15212-1-AP, 1:500, Proteintech Group Inc., Rosemont, IL, USA), mitochondrial complex I (mouse anti-NDUFS3 subunit, ab110246, 1: 2000, Abcam Inc., Waltham, MA, USA), complex II (mouse anti-SDHA subunit, ab14715, 1:2500, Abcam Inc., Waltham, MA, USA), complex III (mouse anti-UQCRC2 subunit, ab14745, 1:1000, Abcam Inc., Waltham, MA, USA), complex IV (rabbit anti-subunit IV, 4844, 1:1000, Cell Signaling Technology, Danvers, MA, USA), ß-actin (ab8227, 1:1000, Abcam Inc., Waltham, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (2174, Cell Signaling Technology, 1:2000, Danvers, MA, USA).

Techniques: Over Expression, Knock-Out

Journal: Biomolecules

Article Title: The Proteomic Landscape of Parkin-Deficient and Parkin-Overexpressing Rat Nucleus Accumbens: An Insight into the Role of Parkin in Methamphetamine Use Disorder

doi: 10.3390/biom15070958

Figure Lengend Snippet: Parkin Deficit or Overexpression in Rat Nucleus Accumbens Changes Krebs Cycle Enzyme Levels in Opposite Directions. SDS-PAGE and western blotting showed statistically significant differences between parkin knockout (PKO) and parkin-overexpressing (PO) rats, as well as between these groups and wild-type (WT) controls. ( A ) dihydrolipoamide dehydrogenase (DLST), ( B ) mitochondrial malate dehydrogenase (MDH2), ( C ) 2-oxoglutarate dehydrogenase (OGDH), ( D ) dihydrolipoyl dehydrogenase (DLD), and ( E ) citrate synthase (CS). One-way ANOVA with Holm-Sidak’s post hoc test). * p < 0.05, ** p < 0.01, *** p < 0.001, n = 7/group. The data is expressed as mean ± SEM. ( F ) CS levels show a positive correlation with parkin levels ( p < 0.05, Pearson correlation test). Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Proteins were transferred to PVDF membrane (EMD Millipore, Burlington, MA, USA) at 400 mA, blocked with 5% non-fat dried milk dissolved in TBST (10 mM Tris, 150 mM NaCl, and 0.05% Tween-20), and incubated overnight at 4 °C with the following primary antibodies (diluted in 5% milk-containing TBST) against the following proteins: citrate synthase (CS) (NBP2-13878, 1:1000 Novus Biologicals, Centennial, CO, USA), dihydrolipoamide S-succinyltransferase (DLST) (PA5-51794, 1:1000,Invitrogen Life Technologies, Carlsbad, USA), mitochondrial malate dehydrogenase (MDH2) (9610, 1:1000, Cell Signaling, Danvers, MA, USA), dihydrolipoamide dehydrogenase (DLD) (NBP1-31302, 1:1000, Novus Biologicals, Centennial, CO, USA), 2-oxoglutarate dehydrogenase (OGDH) (15212-1-AP, 1:500, Proteintech Group Inc., Rosemont, IL, USA), mitochondrial complex I (mouse anti-NDUFS3 subunit, ab110246, 1: 2000, Abcam Inc., Waltham, MA, USA), complex II (mouse anti-SDHA subunit, ab14715, 1:2500, Abcam Inc., Waltham, MA, USA), complex III (mouse anti-UQCRC2 subunit, ab14745, 1:1000, Abcam Inc., Waltham, MA, USA), complex IV (rabbit anti-subunit IV, 4844, 1:1000, Cell Signaling Technology, Danvers, MA, USA), ß-actin (ab8227, 1:1000, Abcam Inc., Waltham, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (2174, Cell Signaling Technology, 1:2000, Danvers, MA, USA).

Techniques: Over Expression, SDS Page, Western Blot, Knock-Out

Journal: Biomedicines

Article Title: Immunological and Structural Characterization of Titin Main Immunogenic Region; I110 Domain Is the Target of Titin Antibodies in Myasthenia Gravis

doi: 10.3390/biomedicines11020449

Figure Lengend Snippet: Schematic representation of the domain composition of titin and fragments used in the present study. ( A ) Domain composition of titin. Fibronectin domains are shown in white, Ig domains in purple and unique sequences in grey. Titin kinase (TK) is in orange. Titin sarcomeric regions and A-band super-repeats are annotated. The first domain of each zone is numbered. Segments studied in this work are underlined. The MIR segment and its constituents studied in this work are shown; ( B ) Amino acid sequence of I110 and its corresponding 9 synthetic peptides in this work (horizontal thin lines under the sequence).

Article Snippet: SL collaborates with DLD Diagnostika Hamburg on titin autoantibody detection by ELISA ( https://www.dld-diagnostika.de/uploads/uploads/Tit-e_4.pdf (accessed on 27 July 2021)).

Techniques: Sequencing

Journal: Biomedicines

Article Title: Immunological and Structural Characterization of Titin Main Immunogenic Region; I110 Domain Is the Target of Titin Antibodies in Myasthenia Gravis

doi: 10.3390/biomedicines11020449

Figure Lengend Snippet: Binding of MG sera to the titin fragments by ELISA. Sera (from 10 MG patients and a healthy control, HC) were incubated with immobilized fragments: whole MIR, its domains I109-I110, I110-I111, I110 and I111, and the titin fragments outside the MIR region: M1-M2, I99-I101, titin kinase region A170-Kin-M1, and A160-A170. ( A ) It is shown that all sera were bound to all 3 fragments which contain the I110 domain, practically equally well with their binding to the whole MIR, but not to any other fragment. ( B ) Average percentages of antibody binding of the 10 sera to I109-I110, I110-I111, and I110, versus the values of antibody binding to the MIR. The differences are not statistically significant.

Article Snippet: SL collaborates with DLD Diagnostika Hamburg on titin autoantibody detection by ELISA ( https://www.dld-diagnostika.de/uploads/uploads/Tit-e_4.pdf (accessed on 27 July 2021)).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Control, Incubation

Journal: Biomedicines

Article Title: Immunological and Structural Characterization of Titin Main Immunogenic Region; I110 Domain Is the Target of Titin Antibodies in Myasthenia Gravis

doi: 10.3390/biomedicines11020449

Figure Lengend Snippet: Efficiency of the I110 domain to block sera from binding to the MIR and MIR fragments. ( A ) Binding of the 10 MG sera of to the four MIR fragments and inhibition of binding by the soluble M1-M2 and I110 fragments. Each serum, before incubation with the immobilized titin fragment (MIR, I109-I110, I110 and I110-I111), was preincubated with the soluble M1-M2 or I110 fragments or with plain buffer (untreated (-)). It is shown that preincubation with I110 blocked all antibodies to all 4 immobilized fragments (all contain I110 domain), whereas the soluble M1-M2 fragment did not affect antibody binding to any of the immobilized fragments. ( B ) Binding of another 20 MG sera (no 11–30) to the MIR and I110 fragments (1st and 2nd columns of each patient), and its inhibition by the soluble I110 fragment (3rd and 4th columns). These data confirm that all anti-MIR antibodies bind to I110 epitopes.

Article Snippet: SL collaborates with DLD Diagnostika Hamburg on titin autoantibody detection by ELISA ( https://www.dld-diagnostika.de/uploads/uploads/Tit-e_4.pdf (accessed on 27 July 2021)).

Techniques: Blocking Assay, Binding Assay, Inhibition, Incubation

Journal: Biomedicines

Article Title: Immunological and Structural Characterization of Titin Main Immunogenic Region; I110 Domain Is the Target of Titin Antibodies in Myasthenia Gravis

doi: 10.3390/biomedicines11020449

Figure Lengend Snippet: Search for correlation of thymoma state, age at onset and gender of MG patients versus the binding pattern to titin fragments. ( A – C ): Sera from 3 groups of MG patients with hyperplastic thymus ( A ), involuted thymus ( B ), or thymoma ( C ), further placed in subgroups of early onset (<40 years) and late onset MG, males or females, were tested for binding to the 9 fragments: MIR, I109-I110, I110-I111, I110, I111, M1-M2, I99-I101, titin kinase region A170-Kin-M1 and polyFn. In each group, first are shown the sera from early onset (<40) MG and then from late onset, and in each subgroup the male patients are placed first, followed by the females (marked with m, f next to patient number). The encircled patient C6 is the only one in the whole study whose serum was bound to I110 only about 47% of its binding to each of the other 3 samples. ( D ) Average percentages of antibody binding of the 3 groups of sera to I109-I110, I110-I111 and I110, versus the values of antibody binding to the MIR. The differences are not statistically significant.

Article Snippet: SL collaborates with DLD Diagnostika Hamburg on titin autoantibody detection by ELISA ( https://www.dld-diagnostika.de/uploads/uploads/Tit-e_4.pdf (accessed on 27 July 2021)).

Techniques: Binding Assay

Journal: Biomedicines

Article Title: Immunological and Structural Characterization of Titin Main Immunogenic Region; I110 Domain Is the Target of Titin Antibodies in Myasthenia Gravis

doi: 10.3390/biomedicines11020449

Figure Lengend Snippet: Binding to titin fragments of sera from patients suspected for paraneoplastic syndromes. Patients’ sera were initially screened for antibodies to 13 paraneoplastic antigens, including titin MIR, by dot blot. All sera which were found positive for titin MIR by dot blot (1st column for each serum shows the intensity of the dot in arbitrary units; right Y -axis) were further tested by ELISA for binding to the 3 MIR fragments: whole MIR, I110 and I111. The 3 solid bars for each patient represent ELISA values for the immobilized fragments: whole MIR, I110 and I111 (left Y -axis). It is shown that a) there is a good correlation between dot blot MIR intensity and ELISA MIR values, with only one exception (patient 4); b) as with the MG sera, binding to the I110 is very similar with that to the MIR, whereas practically no serum was bound to I111.

Article Snippet: SL collaborates with DLD Diagnostika Hamburg on titin autoantibody detection by ELISA ( https://www.dld-diagnostika.de/uploads/uploads/Tit-e_4.pdf (accessed on 27 July 2021)).

Techniques: Binding Assay, Dot Blot, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Immunological and Structural Characterization of Titin Main Immunogenic Region; I110 Domain Is the Target of Titin Antibodies in Myasthenia Gravis

doi: 10.3390/biomedicines11020449

Figure Lengend Snippet: Binding of anti-titin antibodies to denatured I110 domain by Western blotting. ( A ) Titin MIR samples were SDS-electrophoresed and transferred onto polyvinylidene difluoride (PVDF) membranes, which were then incubated with 1/100 dilutions of 3 test MG sera, and a HC serum. It is shown that all MG sera recognized the I110 and the two I110-containing fragments but not the I111 fragment, whereas the HC serum showed no binding to any fragment. ( B ) Five more MG anti-MIR/I110 sera were incubated with blotted I110. ( C ) The incubated “depleted” sera of 6B were then used in ELISA with immobilized I110 fragment and compared with the binding of the corresponding untreated sera. Blue columns: untreated sera; red columns: treated “depleted” sera. Percentages above red columns denote the % of unbound I110 antibodies (after deduction of the background values marked with the dotted line). The average % of unbound antibodies was 33%.

Article Snippet: SL collaborates with DLD Diagnostika Hamburg on titin autoantibody detection by ELISA ( https://www.dld-diagnostika.de/uploads/uploads/Tit-e_4.pdf (accessed on 27 July 2021)).

Techniques: Binding Assay, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: The human type 2 diabetes-specific visceral adipose tissue proteome and transcriptome in obesity

doi: 10.1038/s41598-021-96995-0

Figure Lengend Snippet: Differentially abundant proteins in DM compared with NDM patients.

Article Snippet: Band density values between separate blots were normalized to a 50 μg referent protein sample comprised of a 50:50 mixture of one DM sample and one NDM sample, then normalized by densitometry values between samples to matched β-actin loading controls, using the following antibodies: DLD (Novus Biologicals Inc., Littleton, CO, USA, Cat#NBP2-19361, 1:3000); SDH-α (Thermo Fisher Scientific Inc., Waltham, MA, USA, Cat#PA5-79964, 1:3000); FASN (Cell Signaling Inc., Danvers, MA, USA, Cat#3189S, 1:1000); β-Actin (Invitrogen Inc., Waltham, MA, USA, Cat#MA5-15739, 1:10,000), anti-rabbit HRP (Sigma-Aldrich, St. Louis, MO, USA Cat#AP132P, 1:10,000) and anti-mouse HRP (Thermo Fisher Scientific Inc., Waltham, MA, USA, Cat#31430, 1:10,000).

Techniques: Methylation, Coagulation, Phospho-proteomics, Activity Assay