Journal: Advanced Materials (Deerfield Beach, Fla.)

Article Title: An Ultrapotent, Ultraeconomical, Antifreeze Polypeptide

doi: 10.1002/adma.202420504

Figure Lengend Snippet: Polypeptide structural characterization. A) FTIR traces of t Bu‐E L NCA as compared to (A L E L ) 50 . B) FTIR traces of protecting‐group‐free E L NCA as compared to (A L E L ) 50 . C–E) CD spectra of polypeptides in PBS buffer at 20 °C. Data in C) indicates length dependent helical propensity of (A L E L ) n ; D) reveals that (A L E L ) 50 and (A L K L ) 50 adopt right‐handed α‐helices similar to that of winter flounder AFP1 but (V L E L ) 50 is disordered; and E) indicates that mirror‐image (A D E D ) 50 adopts a left‐handed α‐helix while racemic (A L/D E L/D ) 50 is disordered. F) CD spectra in PBS buffer (average of three scans) of (A L E L ) 50 at 4, 95, and at 4 °C after heating, demonstrating that the structure is thermally reversible and, by comparison to 20 °C data in E, that helical order increases slightly as the solution approaches freezing.

Article Snippet: For the primary antibody, α‐EGFP (rabbit polyclonal) in PBS (Cat. No.: 16118‐T16) was purchased from SinoBiological.

Techniques: Circular Dichroism, Comparison

Journal: Journal of Integrative Agriculture

Article Title: Critical role of cytochrome c1 and its cleavage in porcine reproductive and respiratory syndrome virus nonstructural protein 4-induced cell apoptosis via interaction with nsp4

doi: 10.1016/s2095-3119(17)61670-8

Figure Lengend Snippet: Fig. 1 Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 4 (nsp4) interacts with cytochrome c1 (cyto.c1). A, yeast two hybrid assay. The plasmids pGBKT7-nsp4 and pGADT7-cyto.c1 were co-transformed into the yeast strain Y2HGold and selected on QDO/X/ABA plates. The top and second panels are the respective positive and negative controls. B, HEK 293T cells were transfected to express Flag-cyto.c1, or HA-nsp4 or together. At 24 h post-transfection, the cells were either subjected to direct Western blot analysis or lysed and immunoprecipitated with anti-HA antibodies. The proteins bound to sepharose beads were separated by SDS-PAGE, transferred to PVDF membranes, and probed with the antibodies to Flag and HA. C, the same as B except that nsp4 truncation mutants were used. D, MARC-145 cells were transduced with letiviruses that were expressing GFP or nsp4-GFP in the presence of 8 µg mL–1 polybrene, respectively. The cells were harvested 48 h post-transduction and cell lysates were immunoprecipitated using anti-GFP beads. The proteins bound to sepharose beads were subjected to Western blot analysis using an anti-cytochrome c1 polyclonal antibody or anti-GFP mAb. E, PRRSV nsp4 interacts with cyto.c1 in mammalian cells. Vero cells grown on coverslips in six-well plates were transfected when 60–70% to express the indicated proteins, either alone or pairwise. At 18–24 h post-transfection, the cells were fixed, permeablized, and stained with antibodies to HA and Flag, and examined by confocal microscopy. For double transfections, the merged images are shown at the right.

Article Snippet: Mouse anti-green fluorescent protein (GFP) mAb (66002-1-Ig) and rabbit anti-cytochrome c1 polyclonal antibodies (10242-1-AP) were purchased from Proteintech (Chicago, IL, USA).

Techniques: Virus, Y2H Assay, Transformation Assay, Transfection, Western Blot, Immunoprecipitation, SDS Page, Transduction, Expressing, Staining, Confocal Microscopy

Journal: NPJ Precision Oncology

Article Title: Acetylation of ELF5 suppresses breast cancer progression by promoting its degradation and targeting CCND1

doi: 10.1038/s41698-021-00158-3

Figure Lengend Snippet: a Inhibition of deacetylation and protein synthesis on the protein level of ELF5. HEK293T cells were treated without and with 2 μM TSA and 5 mM NAM in the absence or presence of 20 μM MG132. Endogenous ELF5 was probed by anti-ELF5 Ab. b NAM- and TSA-mediated regulation of the ELF5 ubiquitination. Ubiquitination of Flag-ELF5 expressed in HEK293T cells the absence and presence of NAM + TSA with MG132. c Quantitation of the ubiquitination levels of Flag-ELF5 WT, Flag-ELF5 6KR, and Flag-ELF5 6KQ in HEK293T cells. d , e Half-lives of ELF5 and mutants. HEK293T cells expressing GFP-ELF5-WT, GFP-ELF5-6KR, or GFP-ELF5-6KQ were treated with 50 μM Cycloheximide, and the expression levels of these proteins were analyzed by western blot with anti-GFP Ab. The graph shown represents the ELF5 half-lives in Fig. 5e. Data are the means ± SDs from three determinations. * p < 0.05; ** p < 0.01. f , g The expression of ELF5 and SIRT6 in human breast cancer tissues. Representative images tissue samples from breast cancer patients showing the expression levels of ELF5 and SIRT6 as assessed by immunohistochemistry. Both ELF5 and SIRT6 levels were classified as low, medium, or high based on the intensities of their IHC staining, and the percentages of patients classified in each category are depicted in the histogram in Fig. 5g.

Article Snippet: The primary antibodies were the following: Anti-ELF5 (1:1000 for WB, 1:500 for IP, 1:50 for immunofluorescence, sc-376737, Santa Cruz Biotechnology, CA, USA), anti-ELF5 (1:1000 for WB, 1:500 for IP, sc-166653, Santa Cruz Biotechnology, CA, USA), anti-Fibrillarin (1:3000 for WB, sc-166001, Santa Cruz Biotechnology, CA, USA), anti-β-actin (1:1000 for WB, sc-47778, Santa Cruz Biotechnology, CA, USA), anti-p300 (1:3000 for WB, 1:500 for IP, 1:100 for immunofluorescence, sc-585, Santa Cruz Biotechnology, CA, USA), anti-SIRT6 (1:3000 for WB, 1:500 for IP, ab62739, Abcam, Cambridge, UK), anti-Cyclin E (1:3000 for WB, ab7959, Abcam, Cambridge, UK), anti-Flag ® M2 (1:2000 for WB, F1804, Sigma-Aldrich, Saint Louis, Mo, USA), anti-MYC (1:2000 for WB, PLA0001, Sigma-Aldrich, Saint Louis, Mo, USA), anti-Flag ® (1:2000 for WB, 1:500 for IP, F7425, Sigma-Aldrich, Saint Louis, Mo, USA), anti-GFP (1:2000 for WB, TA150052, Origene, Rockville, MD, USA), anti-Myc (1:2000 for WB, TA150121, Origene, Rockville, MD, USA), anti-GFP (1:3000 for WB, 1:500 for IP, GTX113617, GeneTex, San Antonio, TX, USA), anti-HA (1:3000 for WB, 1:500 for IP, GTX115044, GeneTex, San Antonio, TX, USA), anti-GST (1:2000 for WB, ABN116, Millipore, Boston, MA, USA), anti-acetylated-lysine (1:3000 for WB, 1:500 for IP, #9441, Cell Signaling Technology, Boston, MA, USA).

Techniques: Inhibition, Quantitation Assay, Expressing, Western Blot, Immunohistochemistry

Journal: Scientific Reports

Article Title: Integrins mediate placental extracellular vesicle trafficking to lung and liver in vivo

doi: 10.1038/s41598-021-82752-w

Figure Lengend Snippet: Key resources. Information includes species and strains of animals used in this study, software, kits, antibodies, and reagents.

Article Snippet: Antibody , Anti-EGFP (goat polyclonal) , Origene , Origene:TA150095 , (1:2000).

Techniques: Software, Imaging, Labeling, Isolation, Recombinant, Plasmid Preparation