anti ckap4  (Proteintech)

Journal: PLOS Pathogens

Article Title: Identification and characterization of a ubiquitin E3 RING ligase of the Chlamydia -like bacterium Simkania negevensis

doi: 10.1371/journal.ppat.1013626

Figure Lengend Snippet: (A, B) A FLAG-tagged version of the SneRING was overexpressed in U2OS cells using PEI MAX transfection. 24 h later, cells were infected with Sne at an MOI of 1. Non-transfected cells served as a control. 48 h p.i., samples were collected and immunoprecipitation was performed using FLAG-magnetic beads, followed by mass spectrometry analysis. The graphs show identified proteins, with significance (-log 10 p-value calculated by two-tailed T-test, n = 3) plotted against the log 2 fold change (log 2 FC) of transfected/infected U2OS cells (U2OS+Sne + SneRING) relative to non-transfected/infected controls (U2OS+Sne) (A) or of transfected/infected U2OS cells (U2OS+Sne + SneRING) relative to transfected/non-infected controls (U2OS+SneRING) (B) . Enriched proteins are labeled in red, reduced proteins are shown in blue, and grey represents unchanged proteins. Only host cell proteins are shown. (C) Samples were prepared, and immunoprecipitation was performed as in A. Input and elution fractions were analyzed by SDS-PAGE and western blot using antibodies against Sne heat shock protein SnGroEL (star indicates that the signal represents a cross-reactive band observed at 130 kDa), Mic60, CKAP4, Erlin2, PHB, Cox5a, Tom70, and FLAG. Mic60, mitochondrial contact site and cristae organizing system 60; CKAP4, cytoskeleton‐associated protein 4; Erlin2, ER Lipid Raft Associated 2; PHB, Prohibitin; Cox5a, cytochrome c oxidase subunit 5A; Tom70, translocase of the outer mitochondrial membrane 70.

Article Snippet: The CKAP4 antibody (Cat. No. 16686-1-AP) was purchased from Proteintech (Rosemont, USA).

Techniques: Transfection, Infection, Control, Immunoprecipitation, Magnetic Beads, Mass Spectrometry, Two Tailed Test, Labeling, SDS Page, Western Blot, Membrane

Journal: PLOS Pathogens

Article Title: Identification and characterization of a ubiquitin E3 RING ligase of the Chlamydia -like bacterium Simkania negevensis

doi: 10.1371/journal.ppat.1013626

Figure Lengend Snippet: (A, B) A reaction containing purified recombinant SneRING, recombinant E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme (UbcH5b), ubiquitin, and ATP was mixed with the respective substrate (purified recombinant GST (A) or His-CKAP4 (B) ) for the indicated periods at 37 °C. Reactions without the ligase and/or substrate served as controls. Samples were analyzed by SDS-PAGE and western blot, using antibodies against SneRING, GST, ubiquitin, and CKAP4. Asterisks indicate ubiquitinated CKAP4. Ponceau staining of the western blot membranes is shown for the loading control.

Article Snippet: The CKAP4 antibody (Cat. No. 16686-1-AP) was purchased from Proteintech (Rosemont, USA).

Techniques: Purification, Recombinant, Ubiquitin Proteomics, SDS Page, Western Blot, Staining, Control