pmt/bip/v5-his, a s2 cell expression vector  (Thermo Fisher)

Journal: bioRxiv

Article Title: Togaram Ensures Axial Alignment of the Sperm Neck

doi: 10.64898/2026.04.15.718719

Figure Lengend Snippet: (A) Linear maps of human (top) and Drosophila (bottom) Togaram, Cep104, and CCDC66 depicting functional and structural domains. Domains are as indicated in the color key. (B) Both N– and C-terminally GFP-tagged Cep104 (green) binds Togaram (pink) and CCDC66 (blue). S2 cells were co-transfected with the indicated plasmids and anti-GFP co-IPs were prepared from cell lysates. Western blots of inputs and co-IPs were probed for GFP, Flag, and Myc. Arrow head colors correspond to indicated tagged protein colors. (C) Representative ExM images showing wild-type (left), cep104 null (center), and ccdc66 gRNA (right) Canoe stage spermatids with various alignment phenotypes. Spermatids were labeled for the nucleus (DAPI, blue; dotted line), the CA (Asl, magenta), and the centrioles (Ana1, green). Scale bar: ∼1 μm. (D) Quantification of wild-type (n=150), cep104 null (n=98), and ccdc66 gRNA (n=119) spermatids with various alignment phenotypes. (E) Working model in which Togaram, Cep104, and CCDC66 stabilize sperm head/neck microtubules to preserve a straight head-tail axis during the maintenance phase of spermiogenesis. Red question mark highlights a possible mechanism by which these proteins can directly regulate sperm neck stability independent of regulating microtubules.

Article Snippet: S2 cell expression vectors were generated by the Gateway cloning system (ThermoFisher Scientific) to create vectors with Actin5C promoter and the tags of interest (pAFHW, pAGW, pAMW, pAWG; F = Flag, G = GFP, M = Myc; https://emb.carnegiescience.edu/drosophila-gateway-vector-collection ). pAGW-MCS plasmid, expressing GFP alone was used as GFP control for IP experiments.

Techniques: Functional Assay, Transfection, Western Blot, Labeling

Journal: Biochemical and Biophysical Research Communications

Article Title: Spike protein receptor-binding domains from SARS-CoV-2 variants of interest bind human ACE2 more tightly than the prototype spike protein

doi: 10.1016/j.bbrc.2022.12.011

Figure Lengend Snippet: Modules of WHCV-SP. The 1273-amino acid mature SP consists of S1 (residues 1–685) and S2 (residues 686–1273) subunits. S1 contains sequentially, from the 5′-terminus, a 16-residue signal peptide (SP) and a N-terminal domain that extends from amino acid residues 17–318. Next, a receptor binding domain (RBD) spans residues 319–541, within which is contained a receptor binding module (RBM) at residues 438–506. The S2 subunit (residues 542–1273) contains the TMPRSS cleavage site (CS) at residues 685–686 which is cleaved to liberate S1 prior to the SP entering the cells via S2. Next, a transmembrane spanning domain (TMD) is encompassed by residues 1213–1236, followed by a cytoplasmic region at residues 1237–1273.

Article Snippet: RBD inserts containing a SpeI restriction sites at the 5′ end and a MluI restriction site at its 3′ end were cloned from pcDNA3.1SARS2spike into pMT-puro (pND879). pND879 [pMTpuro-Bip signal peptide-MCS-V5- Mlu1 -(His) 6 ] is an efficient in-house S2-cell expression vector backbone, with the original backbone being from the pMT/BiP/V5-His vector (Invitrogen) and pCoPuro (Invitrogen).

Techniques: Binding Assay