Journal: Protein Science : A Publication of the Protein Society

Article Title: Purification and characterization of recombinant human translation initiation factor eIF3

doi: 10.1002/pro.70388

Figure Lengend Snippet: Schematic representation of eIF3 complex expression and purification protocol. (a) Generation of baculovirus for eIF3 expression. Sf9 cells are used to produce P0 and P1 viral progenies. Cell viability was assessed by trypan blue staining and bright field microscopy (top panels, dark cells correspond to death cells). Infected cells were identified based on YFP expression by fluorescence microscopy (red cells are infected cells). (b) Main protocol for purification of eIF3 complexes from over‐expressed in Hi5 insect cells. (c) Analysis of the integrity of purified eIF3 complex by analytical SEC. Chromatogram of SEC run using a Superose6 3.2/300 column of eIF3 expressed in insect cells and purified. Coomassie‐stained SDS polyacrylamide of the fractions indicate in the chromatogram.

Article Snippet: Sf9 insect cell line (ATCC, CRL‐1711) was used to generate the baculoviruses.

Techniques: Expressing, Purification, Staining, Microscopy, Infection, Fluorescence

Journal: bioRxiv

Article Title: Dimerization of human PARP15 is required for NAD + binding and automodification

doi: 10.64898/2025.12.15.694324

Figure Lengend Snippet: A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In SEC–MALS PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.

Article Snippet: Insect cell line Sf21 was used for the expression of FL protein constructs using Bac-to-Bac® baculovirus expression system (Invitrogen).

Techniques: Activity Assay, Mutagenesis, Hydrolysis Assay, Modification, Expressing, Incubation, In Vitro, Western Blot, Purification, Control