Journal: Frontiers in Endocrinology

Article Title: Quantitative Acetylomics Revealed Acetylation-Mediated Molecular Pathway Network Changes in Human Nonfunctional Pituitary Neuroendocrine Tumors

doi: 10.3389/fendo.2021.753606

Figure Lengend Snippet: MS/MS spectrum of the tryptic peptide. (A) The tryptic peptide ALMDEVVK*ATSR from PGK1. (B) The tryptic peptide TATPQQAQEVHEK*LR from triosephosphate isomerase. K* = acetylated lysine residue.

Article Snippet: The negative control IP experiment was performed with the use of the normal mouse IgG antibody (6 μg; B900620, Proteintech) to replace the anti-PGK1 antibody, which tested the specificity of anti-PGK1 antibody.

Techniques: Tandem Mass Spectroscopy

Journal: Frontiers in Endocrinology

Article Title: Quantitative Acetylomics Revealed Acetylation-Mediated Molecular Pathway Network Changes in Human Nonfunctional Pituitary Neuroendocrine Tumors

doi: 10.3389/fendo.2021.753606

Figure Lengend Snippet: Semiquantitative analysis of acetylated PGK1 between NF-PitNETs and controls. PGK1 in protein samples extracted from NF-PitNET and control tissues was immunoprecipitated (IP) with anti-PGK1 antibody. A negative control immunoprecipitation experiment was performed with the normal mouse IgG antibody but not anti-PGK1 antibody to test the specificity of anti-PGK1 antibody. The IP products (PKG1 and IgG), anti-PGK1 antibodies (Ab), and total protein samples (tumor; control) were simultaneously immunoblotted with anti-acetyl-lysine antibody. T = NF-PitNETs. N = controls. M = markers.

Article Snippet: The negative control IP experiment was performed with the use of the normal mouse IgG antibody (6 μg; B900620, Proteintech) to replace the anti-PGK1 antibody, which tested the specificity of anti-PGK1 antibody.

Techniques: Immunoprecipitation, Negative Control

Journal: EMBO reports

Article Title: C9orf72 protein quality control by UBR5-mediated heterotypic ubiquitin chains.

doi: 10.15252/embr.202255895

Figure Lengend Snippet: Figure 1. Loss of SMCR8 causes proteasomal degradation of C9orf72.

Article Snippet: Primary antibodies BAG6 (Proteintech, 26417-1-AP), Calnexin (Abcam, ab22595), C9orf72 (GeneTex, GTX632041), C9orf72 (Proteintech, 25757-1-AP), GET4 (Proteintech, 27768-1-AP), GFP (Roche, 11814460001), HA (Cell Signaling, 3724S), NRF2 (Abcam, ab62352), PSMC4 (Bethyl Laboratories, A303-850A), SGTA (Proteintech, 11019-2-AP), SMCR8 (Abcam, ab202283), a-tubulin (Abcam, ab7291), Ubiquitin clone FK2 (Enzo Life Sciences, BML-PW8810), Ubiquitin K11 clone 2A3/2E6 (Genentech), Ubiquitin K48 clone Apu2 (Millipore, 05-1307), Ubiquitin K63 clone Apu3-A8 (Genentech), Ubiquitin K11/K48-bispecific (Genentech), UBL4A (Proteintech, 14253-1-AP) and UBR5 (Cell Signaling, 65344) were used for immunoblotting at a dilution of 1:1,000 in 5% milk in Tris-buffered saline with Tween20 (TBS-T).

Techniques:

Journal: EMBO reports

Article Title: C9orf72 protein quality control by UBR5-mediated heterotypic ubiquitin chains.

doi: 10.15252/embr.202255895

Figure Lengend Snippet: Figure 2. C9orf72 abundance is regulated by ubiquitination.

Article Snippet: Primary antibodies BAG6 (Proteintech, 26417-1-AP), Calnexin (Abcam, ab22595), C9orf72 (GeneTex, GTX632041), C9orf72 (Proteintech, 25757-1-AP), GET4 (Proteintech, 27768-1-AP), GFP (Roche, 11814460001), HA (Cell Signaling, 3724S), NRF2 (Abcam, ab62352), PSMC4 (Bethyl Laboratories, A303-850A), SGTA (Proteintech, 11019-2-AP), SMCR8 (Abcam, ab202283), a-tubulin (Abcam, ab7291), Ubiquitin clone FK2 (Enzo Life Sciences, BML-PW8810), Ubiquitin K11 clone 2A3/2E6 (Genentech), Ubiquitin K48 clone Apu2 (Millipore, 05-1307), Ubiquitin K63 clone Apu3-A8 (Genentech), Ubiquitin K11/K48-bispecific (Genentech), UBL4A (Proteintech, 14253-1-AP) and UBR5 (Cell Signaling, 65344) were used for immunoblotting at a dilution of 1:1,000 in 5% milk in Tris-buffered saline with Tween20 (TBS-T).

Techniques: Ubiquitin Proteomics

Journal: EMBO reports

Article Title: C9orf72 protein quality control by UBR5-mediated heterotypic ubiquitin chains.

doi: 10.15252/embr.202255895

Figure Lengend Snippet: Figure 4. C9orf72-binding properties of UBR5 and BAG6.

Article Snippet: Primary antibodies BAG6 (Proteintech, 26417-1-AP), Calnexin (Abcam, ab22595), C9orf72 (GeneTex, GTX632041), C9orf72 (Proteintech, 25757-1-AP), GET4 (Proteintech, 27768-1-AP), GFP (Roche, 11814460001), HA (Cell Signaling, 3724S), NRF2 (Abcam, ab62352), PSMC4 (Bethyl Laboratories, A303-850A), SGTA (Proteintech, 11019-2-AP), SMCR8 (Abcam, ab202283), a-tubulin (Abcam, ab7291), Ubiquitin clone FK2 (Enzo Life Sciences, BML-PW8810), Ubiquitin K11 clone 2A3/2E6 (Genentech), Ubiquitin K48 clone Apu2 (Millipore, 05-1307), Ubiquitin K63 clone Apu3-A8 (Genentech), Ubiquitin K11/K48-bispecific (Genentech), UBL4A (Proteintech, 14253-1-AP) and UBR5 (Cell Signaling, 65344) were used for immunoblotting at a dilution of 1:1,000 in 5% milk in Tris-buffered saline with Tween20 (TBS-T).

Techniques: Binding Assay

Journal: EMBO reports

Article Title: C9orf72 protein quality control by UBR5-mediated heterotypic ubiquitin chains.

doi: 10.15252/embr.202255895

Figure Lengend Snippet: Figure 5. Depletion of UBR5 reduces K11/48 ubiquitination of C9orf72 and increases C9orf72 abundance.

Article Snippet: Primary antibodies BAG6 (Proteintech, 26417-1-AP), Calnexin (Abcam, ab22595), C9orf72 (GeneTex, GTX632041), C9orf72 (Proteintech, 25757-1-AP), GET4 (Proteintech, 27768-1-AP), GFP (Roche, 11814460001), HA (Cell Signaling, 3724S), NRF2 (Abcam, ab62352), PSMC4 (Bethyl Laboratories, A303-850A), SGTA (Proteintech, 11019-2-AP), SMCR8 (Abcam, ab202283), a-tubulin (Abcam, ab7291), Ubiquitin clone FK2 (Enzo Life Sciences, BML-PW8810), Ubiquitin K11 clone 2A3/2E6 (Genentech), Ubiquitin K48 clone Apu2 (Millipore, 05-1307), Ubiquitin K63 clone Apu3-A8 (Genentech), Ubiquitin K11/K48-bispecific (Genentech), UBL4A (Proteintech, 14253-1-AP) and UBR5 (Cell Signaling, 65344) were used for immunoblotting at a dilution of 1:1,000 in 5% milk in Tris-buffered saline with Tween20 (TBS-T).

Techniques: Ubiquitin Proteomics

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Scheme of introduced loxP sites in Orc 2 locus. ( B ) Representative picture of genotyping of offspring coming from Orc2 f/+ crossed with Orc2 f/+ . ( C ) The ratio of observed to expected animals coming from Orc2 f/+ crossed with Orc2 f/+ . ( D ) Schematic of the ORC2 protein and the DeltaORC2 protein produced after deletion of exons 6 and 7. A110 is mutated to V110 and then the protein goes out of frame. ( E ) Validation of Orc2 deletion 3 d after Adeno cre transduction. ( F ) Western blot of ORC2 protein 5 d after Adeno cre transduction. 10 or indicated μl of lysate loaded/lane as written on the top. ( G ) MTT assay of WT and Orc2 f/f MEFs without and with Adeno cre transduction. ( H ) Western blot of ORC2 protein 5 and 15 d after Adeno Cre transduction. Figure 1—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 1—source data 2. Original image for , panel B. Figure 1—source data 3. PDF file containing original DNA gel picture corresponding to , panel E, indicating the relevant bands and increasing Adeno-Cre. Figure 1—source data 4. Original image for , panel E. Figure 1—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel F, indicating the relevant bands and addition of Adeno-Cre. Figure 1—source data 6. Original image for , panel F. Figure 1—source data 7. PDF file containing original Western blot membrane picture corresponding to , panel H, indicating the relevant bands and ORC2 protein expression. Figure 1—source data 8. Original image for , panel H.

Article Snippet: The antibodies used in this study are listed: ORC1 (Santa Cruz; sc-28741); ORC3 (Santa Cruz; sc-374231); ORC5 (Boster Biological technology; A03676-1); ORC6 (Santa Cruz; sc-390490); CDC6 (Santa Cruz; sc-9964); MCM2 (Abcam; ab4461); MCM3 (Santa Cruz; sc-9850).

Techniques: Produced, Biomarker Discovery, Transduction, Western Blot, MTT Assay, Membrane, Expressing

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Scheme of Alb +/- - Orc2 f/f ROSA26 stop-EYFP crossed with Alb +/- -Orc2 f/f ROSA26 stop-EYFP (All mice are with ROSA26 stop-EYFP and so we do not include this in the genotypes below). ( B ) The ratio of observed to expected animals coming from A. ( C ) Western blot of hepatocytes from Orc2 f/f and Alb +/- - Orc2 f/f animals. Tubulin was used as loading control. ( D ) Quantification of the Western blots of hepatocyte lysates from Orc2 f/f (without Alb-cre ) mice and the same genotype but with Alb-Cre to show the levels of other key replication initiation proteins in the ORC2 KO hepatocytes. ( E ) Average body weight of Orc2 f/f and Alb-Orc2 f/f animals. ( F ) Average liver weight of Orc2 f/f and Alb-Orc2 f/f animals. ( G ) Average liver-to-body weight ratio of Orc2 f/f and Alb-Orc2 f/f animals. ( H ) Representative H&E staining of liver tissue from Orc2 f/f (WT) and Alb-Orc2 f/f (KO) animals. Both panels at same scale. ( I ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f animals. ( J ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f female mice. ( K ) Quantification of hepatocytes nuclear size in Orc2 f/f and Alb-Orc2 f/f male mice. *p<0.05, **p<0.01, two-tailed Student’s t-test. Figure 2—source data 1. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent ORC2 protein. Figure 2—source data 2. Original image for , panel C. Figure 2—source data 3. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent Tubulin protein. Figure 2—source data 4. Original image for , panel C.

Article Snippet: The antibodies used in this study are listed: ORC1 (Santa Cruz; sc-28741); ORC3 (Santa Cruz; sc-374231); ORC5 (Boster Biological technology; A03676-1); ORC6 (Santa Cruz; sc-390490); CDC6 (Santa Cruz; sc-9964); MCM2 (Abcam; ab4461); MCM3 (Santa Cruz; sc-9850).

Techniques: Western Blot, Control, Staining, Two Tailed Test, Membrane, Molecular Weight, Labeling

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A–B ) Breeding schemes to obtain conditional double flox animals. ( C ) The ratio of observed to expected animals coming from B. Orc1 =all animals with Orc1 f/f ROSA26 stop-EYFP , Orc2 =all animals with Orc2 f/f ROSA26 stop-EYFP , Orc1 Orc2 =all animals with Orc1 f/f Orc2 f/f ROSA26 stop-EYFP genotype. This was before the introduction of Alb-Cre . ( D ) Immunoblot of hepatocytes from WT ( Orc1 f/f Orc2 f/f ) and DKO ( Orc1 f/f Orc2 f/f Alb-cre +/- ) mice to show that ORC1 and ORC2 are depleted in the DKO cells. ( E ) Quantitation of immunoblots to show that levels of other key initiation protein subunits are not decreased in the DKO mice hepatocytes. ( F ) Average body, liver weight, and their ratio for WT and DKO animals. ( G ) Representative H&E staining of liver tissue from male WT and DKO animals. ( H ) Quantification of hepatocyte nuclear size in the WT and DKO animals. ( I ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells from DKO mice. Figure 6—source data 1. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC1 protein expression, and individual animals. Figure 6—source data 2. Original image for panel D, ORC1 protein expression, and individual animals. Figure 6—source data 3. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC2 protein expression, and individual animals. Figure 6—source data 4. Original image for panel D, ORC2 protein expression, and individual animals. Figure 6—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, HSP90 protein expression, and individual animals. Figure 6—source data 6. Original image for panel D, HSP90 protein expression, and individual animals.

Article Snippet: The antibodies used in this study are listed: ORC1 (Santa Cruz; sc-28741); ORC3 (Santa Cruz; sc-374231); ORC5 (Boster Biological technology; A03676-1); ORC6 (Santa Cruz; sc-390490); CDC6 (Santa Cruz; sc-9964); MCM2 (Abcam; ab4461); MCM3 (Santa Cruz; sc-9850).

Techniques: Western Blot, Quantitation Assay, Staining, Membrane, Expressing

Journal: PLoS Pathogens

Article Title: Anthrax toxin component, Protective Antigen, protects insects from bacterial infections

doi: 10.1371/journal.ppat.1008836

Figure Lengend Snippet: (A) A model of PA 20 -mediated phenomenon. During mammalian anthrax infection (left panel), anthrax PA 83 binds to host CMG2 and TEM8 cell surface receptors. PA 83 is then cleaved by cell surface mammalian furin proteases, yielding PA 20 and PA 63 , with the latter remaining attached to the host cell receptor. PA 63 then multimerizes and binds to the circulating LF or EF molecules. This PA pre-pore is endocytosed with the help of clathrin. Upon entry into the cell, the PA pre-pore becomes the PA-pore. This change allows LF and EF to escape into the cell cytosol. Once in the cytosol, EF functions as an adenyl cyclase resulting in edema. Conversely, LF cleaves MAPKK and Nlrp1, causing caspase-mediated pyroptosis. Upon contact with anthrax infected animals or their carcasses, scavenging insects are likely exposed to PA 20 . PA 20 , once ingested by Drosophila , may interact with the Toll pathway to reduce the sensitivity of flies to bacterial challenge (center panel). Two pathways trigger the expression of antimicrobial peptides in Drosophila : the Toll and Imd pathways. During the bacterial challenge, Toll is activated by bacterial lys-type peptidoglycan (PGN) by binding to the PGRP-SA/GNBP1 heterodimer and undergoing enzymatic modification. This results in Toll activation by SPZ ligand binding. Concurrently or separately, Toll is also known to be activated by the circulating virulence factor detection protein, Psh, which also works through SPZ to induce Toll activation. The Imd pathway responds primarily to bacterial dap-type peptidoglycans such as that found in B . cereus and S . liquefaciens . Both Toll and Imd ultimately activate NF-κB transcription factors, which induce the transcription of AMPs that act in a broad-spectrum manner. PA 20 , once ingested by Drosophila , works through Toll and likely PGRP-SA to reduce the sensitivity of flies to bacterial challenge. The effect of PA 20 on the Toll/NF-κB pathway may also occur on the homologous mammalian pathway (right panel). The vaccination with PA 83 and human cell exposures to PA 83 or PA 20 are known to induce the Toll-like receptors-mediated activation of NF-κB and inflammatory cytokines, such as Interleukins. (B-C) ELISA assays showing the interaction between D . melanogaster PGRP-SA (B) or human PGRP-S (C) with PA 20 or peptidoglycans (PGN). PGRP-SA or PGRP-S are added to wells with immobilized PA 20 or PGNs. Anti-T7 antibody is used to detect T7-tagged PGRP-SA, and anti-PGRP-S antibody is used to detect PGRP-S. (D) PGRP-S pull-down assay. PGRP-S binds to and co-precipitates with the partially soluble PA 20 or the insoluble B . subtilis peptidoglycan (PGN). The supernatant (s) or pellet (p) samples from the pull-down assay were analyzed by the Western blot using anti-PGRP-S (top) or anti-PA (bottom) polyclonal antibodies. (E) The effect of PA 20 on PGRP-SA mutant flies. Drosophila mutants were challenged with B . cereus with or without PA 20 .

Article Snippet: Chemical/reagents (source, catalog #) are: cortisone acetate (TCI, C0389), human recombinant PGRP-S (also called PGLYRP1) (BioVendor, RD172316100), polyclonal mouse anti-PGRP-S antibody (Abnova, H00008993-B01P), monoclonal mouse Anti-T7 tag antibody (Sigma-Aldrich, T8823), peptidoglycan from B . subtilis and Staphylococcus aureus (Sigma-Aldrich, 69554 and 77140, respectively), polyclonal goat anti-PA antibodies (List Biological Laboratories, 771B), rabbit anti-goat-HRP secondary antibody (Invitrogen, 81–1620), and goat anti-mouse-HRP secondary antibody (Bio-Rad, 172–1011).

Techniques: Infection, Expressing, Binding Assay, Modification, Activation Assay, Ligand Binding Assay, Enzyme-linked Immunosorbent Assay, Pull Down Assay, Western Blot, Mutagenesis

Journal: Aging (Albany NY)

Article Title: PGC-1α activator ZLN005 promotes maturation of cardiomyocytes derived from human embryonic stem cells

doi: 10.18632/aging.103088

Figure Lengend Snippet: The expression of PGC-1α was upregulated during cardiomyocyte differentiation; ZLN005 increased PGC-1α mRNA and protein level in hESC-CMs. ( A ) The relative mRNA and ( B ) protein expression of PGC-α during cardiomyocyte differentiation (mRNA, n=7; protein, n=5). ( C ) Schematic representation of the experimental schedule including hESC culture, cardiomyocyte differentiation, culture and treatment. ( D ) Effect of ZLN005 on mRNA levels (n=6). ( E ) Effect of ZLN005 on PGC-α protein expression (n=12).

Article Snippet: The primary antibodies were as follows: anti-PGC-1α antibody (1:500, Boster biological), Connexin 43 antibody (1:400, Boster biological), and anti-GAPDH antibody (1:5000, Abcam).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: PGC-1α activator ZLN005 promotes maturation of cardiomyocytes derived from human embryonic stem cells

doi: 10.18632/aging.103088

Figure Lengend Snippet: The used primers in the study.

Article Snippet: The primary antibodies were as follows: anti-PGC-1α antibody (1:500, Boster biological), Connexin 43 antibody (1:400, Boster biological), and anti-GAPDH antibody (1:5000, Abcam).

Techniques: Sequencing