Journal: The Journal of Cell Biology

Article Title: Identification of Ubiquilin, a Novel Presenilin Interactor That Increases Presenilin Protein Accumulation

doi:

Figure Lengend Snippet: Schematic drawings of ubiquilin expression constructs. (I) The full-length ubiquilin polypeptide consists of 595 residues and contains an NH 2 -terminal UB domain (speckled), a COOH-terminal UBA domain (striped), and several regularly spaced asparagine-proline (Asn-Pro) repeats (vertical bars). (II) The probes used in human Northern blots. (III) GST-fusion constructs: A (N393–S595 aa), B (Q378–S595 aa), C (Q113–M377 aa), D (Q541–S595 aa), E (D449–S595 aa), F (D449–L540 aa), G (N393–L540 aa), H (M37–S595 aa), I (M37–L540 aa), J (Q113–L540 aa), K (Q113–S595 aa), and L (GST alone). The ubiquilin portions of constructs A and B were isolated in the original Y2H screen. Bacterially expressed GST–fusion B and C polypeptides were used as immunogens for anti-ubiquilin pAb production (*). (IV) Mammalian expression constructs: M, full-length untagged ubiquilin; N, NH 2 -terminal GFP-tagged ubiquilin fused at residue 20 (Ala); and O, COOH-terminal myc epitope-tagged ubiquilin.

Article Snippet: Rabbit anti-ubiquilin polyclonal antisera were generated against purified GST–ubiquilin fusion proteins B or C ( III) by Covance Research Products, Inc. Affinity-purified anti–ubiquilin-C antibodies were obtained by passing anti–C-serum first over a column containing GST alone coupled to CH-Sepharose 4B beads at 4°C overnight, to eliminate antibodies binding to GST, and then by mixing the flow-through fraction with ubiquilin–coupled CH-Sepharose 4B beads at 4°C overnight.

Techniques: Expressing, Construct, Northern Blot, Isolation, Residue

Journal: The Journal of Cell Biology

Article Title: Identification of Ubiquilin, a Novel Presenilin Interactor That Increases Presenilin Protein Accumulation

doi:

Figure Lengend Snippet: Ubiquilin binds presenilins in vitro. (A and B) GST pull-down experiments. Full-length in vitro synthesized 35 S-labeled PS2 and PS1 (first lanes) migrated in SDS-PAGE gels with broad bands of 54 and 48 kD (arrowheads), respectively, along with a smear of slower migrating forms, presumably due to the highly hydrophobic nature of the proteins. [ 35 S]PS complexes (especially the slower migrating forms) were retained by GST–ubiquilin constructs containing the UBA domain (lane letters correspond to constructs shown in III), but not by those lacking the domain, or by GST alone. (C) Immunoprecipitation experiments. PS2-transfected HeLa cell lysates were immunoprecipitated with preimmune sera or corresponding anti–PS2-Loop antibody and anti-PS2 NH 2 terminus antibody. After separation by SDS-PAGE, coprecipitating ubiquilin (arrowhead) was detected by immunoblotting with anti–ubiquilin-B antibody. (D–F) Cell fractionation experiments. Parallel immunoblots of equal portions of soluble supernatant (S) and insoluble pellet (P) HeLa cellular fractions prepared without the use of detergent (−) or in the presence of 1% Triton X-100 (+) with (D and F) anti-ubiquilin or (E) anti-PS2 antibodies. The HeLa cells used for cell fractionation in D were untransfected, whereas, in E and F, the cells were transfected with a full-length wild-type PS2 construct. The relative ratio of ubiquilin protein in the P− compared with the S− fractions was determined by densitometric analysis of the autoradiographs. This analysis indicated that transfection of presenilin caused 30% more (F) ubiquilin protein to partition in the pellet fraction in the absence of detergent compared with (D) untransfected cells. The partitioning of lamin A/C proteins after cell fractionation from untransfected and PS2-transfected cells was monitored with anti-lamin A/C antibodies and shown below in D and F, respectively.

Article Snippet: Rabbit anti-ubiquilin polyclonal antisera were generated against purified GST–ubiquilin fusion proteins B or C ( III) by Covance Research Products, Inc. Affinity-purified anti–ubiquilin-C antibodies were obtained by passing anti–C-serum first over a column containing GST alone coupled to CH-Sepharose 4B beads at 4°C overnight, to eliminate antibodies binding to GST, and then by mixing the flow-through fraction with ubiquilin–coupled CH-Sepharose 4B beads at 4°C overnight.

Techniques: In Vitro, Synthesized, Labeling, SDS Page, Construct, Immunoprecipitation, Transfection, Western Blot, Cell Fractionation

Journal: Cureus

Article Title: Real-World Evaluation of the Safety and Efficacy of Surgi-ORC® in Orthopedic Surgeries: A Multicenter Prospective Study

doi: 10.7759/cureus.78616

Figure Lengend Snippet: Comparison of mean time to hemostasis (TTH) across different product types and surgery type

Article Snippet: Surgi-ORC® Fibril consists of seven layers of fibrillose material that guarantee thorough integration and absorption throughout the body.

Techniques: Comparison