flag ggs x3 apex2 tagged lentiviral plasmids  (Addgene inc)

Journal: bioRxiv

Article Title: Discovery and CryoEM Structure of FPM13, a Periplasmic Metalloprotein Unique to Francisella

doi: 10.1101/2025.05.08.652791

Figure Lengend Snippet: Genomic organization and expression of F. novicida strains with ALFA tagged FPM13 and FLAG tagged APEX2 with or without a signal sequence (SS) for secretion. (A) Gene cassette of FLAG-tagged APEX2 with or without the first 24 amino acids of the F. novicida beta-lactamase (BlaB) driven by the promoter of F. tularensis LVS bacterioferritin (bfr) was inserted into the blaB locus. The cleavage site of the BlaB signal peptide sequence is indicated with a downward arrow. (B) Proteins from strains grown in TSBC (lanes 2, 3) or TSBC containing 5% KCl (lanes 4, 5) were separated on SDS-PAGE and the protein profiles imaged by stain free UV imaging (left panel). The proteins were transblotted onto nitrocellulose membrane and probed with FLAG-HRP to detect the expression of APEX2 and ssAPEX2 (middle panel) or with ALFA-HRP to detect the expression of FPM13 (right panel). Lane 1, molecular mass standards.

Article Snippet: APEX2 sequence was amplified by PCR from Vimentin - APEX2 in pECFP, a gift from Alice Ting (Addgene plasmid #66170).

Techniques: Expressing, Sequencing, SDS Page, Staining, Imaging, Membrane

Journal: bioRxiv

Article Title: Discovery and CryoEM Structure of FPM13, a Periplasmic Metalloprotein Unique to Francisella

doi: 10.1101/2025.05.08.652791

Figure Lengend Snippet: (A) FPM13 is biotinylated in the presence of H 2 O 2 and pulled down by streptavidin-agarose APEX2 that is targeted to the periplasm (ssAPEX2), but not by cytosolic APEX2. GroEL and KatG serve as cytosolic and periplasmic control proteins, respectively. (B - C) TX-114 phase extraction shows that FPM13 and KatG are soluble proteins that partition into the aqueous phase, unlike the known lipoprotein, IglE (C, lane 4). FPM13-ALFA is not secreted into the culture filtrate (C, lane5), unlike positive control IglC ( D , lane 6). Stain free UV-image shows equal loading of lanes being compared (E).

Article Snippet: APEX2 sequence was amplified by PCR from Vimentin - APEX2 in pECFP, a gift from Alice Ting (Addgene plasmid #66170).

Techniques: Control, Extraction, Positive Control, Staining

Journal: Journal of Virology

Article Title: The ACBD3 protein coordinates ER-Golgi contacts to enable productive TBEV infection

doi: 10.1128/jvi.02224-24

Figure Lengend Snippet: Identification of ACBD3 as a host factor in NS4B-APEX2 proximal protein analysis. ( A ) Confocal fluorescence micrographs of HEK293T cells transiently expressing TBEV NS4B-GFP infected with LGTV (MOI 10, 16 h.p.i.) and stained with anti-calnexin (CANX, ER marker) antibodies and anti-dsRNA antibodies. Scale bar, 10 µm. ( B ) Schematic illustration of the NS4B-APEX2 proximal protein biotinylation screen used in this study. ( C ) Volcano plots of the proteins identified in the NS4B-APEX2 proximal protein biotinylation screen in NS4B-APEX2 cells (left) and NS4B-APEX2 cells infected with LGTV (MOI 10, 16 h.p.i.) (right). Proteins are indicated by dots color-coded based on the FC and P -values. Three biological replicates per condition were analyzed, and the P -value was calculated using unpaired t -test. ( D ) Schematic illustration of ERES-Golgi contacts, and the top hit proteins ACBD3, TFG, SEC23IP, and KTN1 ( E ) Quantification of the percentage of GFP-positive KD HEK293T cells expressing E protein at 24 h after LGTV infection (MOI 1). The KD proteins are indicated. Mean ± SD of 3 independent experiments. One-way ANOVA with Dunnett multiple tests, *** P < 0.005, **** P < 0.0001.

Article Snippet: The coding sequence of APEX2 was PCR-amplified from APEX2 plasmid (a gift from Alice Ting, Addgene plasmid #72480, [ ]) using primers containing restriction sites NotI (5’) and XhoI (3’).

Techniques: Fluorescence, Expressing, Infection, Staining, Marker