Journal: PLOS ONE

Article Title: Reversion from basal histone H4 hypoacetylation at the replication fork increases DNA damage in FANCA deficient cells

doi: 10.1371/journal.pone.0298032

Figure Lengend Snippet: Antibodies.

Article Snippet: Anti-PCNA Antibody (Ms) , Novus Biologics , Cat# NB500-106 RRID:AB_2252058.

Techniques:

Journal: bioRxiv

Article Title: Orally Delivered Milk-Derived Nanovesicles Loaded with Connexin 43 Peptides for Targeted Cardiac Ischemia-Reperfusion Therapy

doi: 10.1101/2025.01.01.630994

Figure Lengend Snippet: A) Schematic of mEV isolation protocol showing the steps of the procedure. Fractions referred to are those collected during Sepharose Column (SEC) fractionation. Fractions 7 through 9 (F7-F9) show highest levels of mEV enrichment and samples used in this study were pooled from F7-F9. Fractions >14 show enrichment for contaminating milk proteins such as casein, with low or no evidence of mEVs, and are typically discarded. B) Single Molecule Localization Microscopy (SMLM) image of an isolated mEV isolated by the protocol in (A) tagged for CD81 (red) and CD63 (teal). C) Negative stain transmission electron microscopy (TEM) indicating dense accumulations of mEVs generated by the isolation protocol. D) Calcein AM loading assay illustrating membrane intactness of isolated mEVs E) Western blotting of mEV isolates for CD81, CD9, TSG-101, and negativity and markedly reduced expression of Calnexin and Casein, respectively. Lysates from HeLa cells are included as a control. Sepharose fraction 16 (F16) shows elevated casein, but no evidence of EV markers. F) Nanoparticle Tracking Analysis (NTA) of an mEV isolate indicating >5×10 12 mEVs per mL, with average nanoparticle size of 125.0 nm, consistent with small EVs. G) SMLM-generated size distribution of CD9/CD81/CD63-positive mEVs, showing a similar histogram morphology to NTA results in panel F.

Article Snippet: Overnight primary antibody incubation was performed and primary antibodies were diluted in the blocking buffer as follows: CD81 (Cell Signaling Technology, 56039S, 1:1000) CD9 (Novus Biologicals, NB500-494, 1:1000), Calnexin (MilliporeSigma, AB2301, 1:5000), TSG101 (Bethyl Laboratories Inc. A303-506A, 1:5000), Casein (Abcam, Ab166596, 1:2000), Cx43 Cytoplasmic-Loop (Invitrogen, PA5-11632, 1:1000), and antibodies against the Cx43 CT (MilliporeSigma, C6219, 1:4000 and Abcam, Ab87645, 1:1000).

Techniques: Isolation, Fractionation, Microscopy, Staining, Transmission Assay, Electron Microscopy, Generated, Membrane, Western Blot, Expressing, Control

Journal: Viruses

Article Title: Reduced-Beclin1-Expressing Mice Infected with Zika-R103451 and Viral-Associated Pathology during Pregnancy

doi: 10.3390/v12060608

Figure Lengend Snippet: ZIKV infection in Becn1 +/+ and Becn1 +/− pregnant dams. ( A ) Schematic diagram illustrating Zika virus (ZIKV)-infection in timed-pregnant dams. Prior to viral infection, pregnant dams received the antibody MAR1-5A3 at 2 mg/animal via intraperitoneal (ip) route at gestational day 8 followed by subcutaneous (sc) infection with ZIKV at 10 3 plaque-forming unit (PFU) in 50 µL of PBS or mock (PBS) injection at gestational day E9. ( B ) Representative Western Blots probed with antibodies against several autophagy proteins, and B-actin was used as an internal control. Adult Becn1 +/+ and Becn1 +/− brains were removed postmortem and minced according to the Materials and Methods. ( C ) Densitometric analysis using image J indicate the levels of p62, Beclin1, ATG5, LC3-I and LC3-II in brains of adult Becn1 +/+ (black bar) and Becn1 +/− (brown bar) mice. The error bars show mean ± SEM for N = 3 animals per treatment. The data were analyzed using GraphPad Prism and two-way analysis of variance (ANOVA) followed by Tukey’s test. * p < 0.05 and ** p < 0.01 vs. Becn1 +/+ . ( D ) Weight gain, expressed in grams, was measured using an analytical balance at gestation day 0 and throughout gestation period, at 3-day intervals. ( E ) Percent survival rate in pregnant dams infected with ZIKV or mock (PBS) was calculated by dividing the total number of live animals by the number of live + dead animals X 100. ( F ) Viral RNA detected in serum collected from ZIKV-infected dams on E13. ( G ) Viral RNA detected in organs removed postmortem from ZIKV-infected dams on E17. ( D – G ) Error bars show mean ± SEM for N = 5–8 animals per treatment. The data were analyzed using GraphPad Prism and two-way ANOVA followed by Tukey’s test. * p < 0.05 vs. Becn1 +/+ . ( F , G ) Viral RNA equivalent is expressed on a log10 scale after comparison with a standard curve produced using serial 10-fold dilutions of ZIKV RNA from known quantities of infectious virus.

Article Snippet: Immunoblots were labeled with primary antibodies against Beclin1 (Catalog# NB500–249), ATG5 (Catalog# NB110-53818), LC3-B (Catalog# NB600–1384) and P62/SQSTM1 (Catalog# NBP1–48320) purchased from Novus Biologicals (Centennial, CO, USA). β-actin (Catalog# sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as internal control.

Techniques: Infection, Virus, Injection, Western Blot, Control, Mass Measurement, Comparison, Produced

Journal: Viruses

Article Title: Reduced-Beclin1-Expressing Mice Infected with Zika-R103451 and Viral-Associated Pathology during Pregnancy

doi: 10.3390/v12060608

Figure Lengend Snippet: Potential link between ZIKV proteins and Beclin1 protein. ( A – D ) The secretions of pro-inflammatory molecules were detected in glial supernatants exposed to 50 nM of viral proteins after 8, 24- and 96-h by ELISA. Becn1 +/+ glia (black bar) and Becn1 +/− glia (brown bar). The data were analyzed by two-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05 vs. respective media control, # p < 0.05 vs. Becn1 +/+ .

Article Snippet: Immunoblots were labeled with primary antibodies against Beclin1 (Catalog# NB500–249), ATG5 (Catalog# NB110-53818), LC3-B (Catalog# NB600–1384) and P62/SQSTM1 (Catalog# NBP1–48320) purchased from Novus Biologicals (Centennial, CO, USA). β-actin (Catalog# sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as internal control.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control