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