Journal: PLoS Pathogens

Article Title: Mechanisms of Assembly and Cellular Interactions for the Bacterial Genotoxin CDT

doi: 10.1371/journal.ppat.0010028

Figure Lengend Snippet: Immunoprecipitation of CDT Complexes Containing Deletion Mutants of CdtA (A) The complexes (10 μg each) were immunoprecipitated with 10 μg anti-myc antibody (myc tag was fused to C-terminus of CdtC) in 150 mM NaCl, 20 mM HEPES (pH 7.5), 0.1% Nonidet P40 substitute, followed by exposure to protein G Sepharose beads, and subjected to nondenaturing SDS-PAGE. (B) Nonspecific binding of CDT complexes to protein G Sepharose was not detected under the same experimental conditions. A, CdtA; B, CdtB; C, CdtC; A*, truncated CdtA; A1, holotoxin with CdtA Δ18−56; A2, holotoxin with CdtA Δ18−67; A3, holotoxin with CdtA Δ18−75; WT, wild-type; Ab, only anti- myc antibody, no CDT; IgG (H+L), nonreduced immunoglobulin G; IgG H, immunoglobulin G heavy chain; IgG L, immunoglobulin G light chain; SN, supernatant.

Article Snippet: Then, 10 μg of protein complex was incubated with 10 μg of antibody in binding buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 0.1% Nonidet P40 Substitute [Sigma, St. Louis, Missouri, United States]) for 3 hr at 4 °C.

Techniques: Immunoprecipitation, SDS Page, Binding Assay

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Refined Immunochemical Characterization in Healthy Dog Skin of the Epidermal Cornification Proteins, Filaggrin, and Corneodesmosin

doi: 10.1369/0022155418798807

Figure Lengend Snippet: Western blotting of dog epidermis extracts. Beagle (Bea) and golden (Gol) retriever dog epidermis was sequentially extracted in Tris-EDTA buffer containing either Nonidet-P40 (TE-NP40 extracts, NP) or 8 M urea (TE-U extracts, U). The extracted proteins were separated by SDS-PAGE, Coomassie blue stained, or immunodetected with AHF10 and G36-19 mAbs, as indicated. The immunoblot corresponding to AHF10 reactivity on the extracts of the beagle dog epidermis was exposed for a longer time (NP′ and U′) to highlight the absence of FLGa-corresponding band. The migration of molecular mass markers (m) is indicated on the left in kDa. ProFLG (Pro) and FLG monomers (FLGa and FLGb) are indicated by arrows, as well as the entire CDSN and its proteolytically derived fragments. For comparison, the Western blotting with AHF10 of TE-NP40 and TE-U extracts of human epidermis (Hum) is shown. Abbreviations: AHF, anti-human filaggrin; PAGE, polyacrylamide gel electrophoresis; FLG, Filaggrin; CDSN, corneodesmosin.

Article Snippet: In brief, human and dog epidermal proteins were sequentially extracted in Tris-HCl pH 7.4 buffer containing nonidet-P40 detergent (Sigma-Aldrich) and EDTA (TE-NP40 extracts), then in TE buffer containing 8 M urea (TE-U extracts), as previously reported.

Techniques: Western Blot, SDS Page, Staining, Migration, Derivative Assay, Polyacrylamide Gel Electrophoresis

Journal: The Journal of Experimental Medicine

Article Title: The Modulation of CD40 Ligand Signaling by Transmembrane CD28 Splice Variant in Human T Cells

doi: 10.1084/jem.20031705

Figure Lengend Snippet: (a) Tyrosine phosphorylation of CD28i induced by CD40L stimulation. CD28-HA–transfected D1.1 cells were serum starved for 6 h and incubated with anti-CD40L Ab for 30 min on ice. Anti–goat IgG was added to start stimulation at 37°C for 2, 5, and 10 min. Nonidet P-40 cell lysates of the stimulated cells were immunoprecipitated with anti-HA Ab. Immunoprecipitates were analyzed on Western blots. The top panel was blotted with anti-Phosphotyrosine (RC20; indicated as p-Tyr). The same membrane was reprobed with anti-HA Ab (indicated as CD28i-HA). L, Ig light chain; lane 1, nonstimulated control; lane 2, CD40L stimulated for 2 min; lane 3, CD40L stimulated for 5 min; lane 4, CD40L stimulated for 10 min. (b) Induction of CD28i-HA expression by doxicyclin in D1.1 transfectant cells. CD28i-HA (∼23 kD) was expressed in D1.1 cells by doxicyclin-inducible promoter and whole cell lysates were assayed by HA-specific Western blotting. (−), doxicyclin-nontreated cells; (+), doxicyclin-treated cells (0.5 μg/ml for 48 h). (c) Overexpression of CD28i-HA enhances the activation of JNK and PAK2 in CD40L-stimulated D1.1 cells. Cells were stimulated with anti-CD40L Ab for periods of time indicated on top. Whole cell lysates were characterized with Western blotting specific for active-form JNK (represented by p46), PAK2 (∼64 kD), or Akt (∼60 kD), and indicated as pJNK, pPAK2, and pAkt on the left of each panel. To measure the level of proteins, the assay membranes were reprobed with JNK-, PAK-, or Akt-specific Abs and indicated as JNK, PAK2, or Akt on the left of each panel. Dox (−), cells not treated with doxicyclin; Dox (+), cells treated with doxicyclin.

Article Snippet: Cell extracts were prepared using lysis buffer containing 1% Nonidet P-40 (BDH Chemicals) or 1% digitonin (Sigma-Aldrich; reference ).

Techniques: Transfection, Incubation, Immunoprecipitation, Western Blot, Expressing, Over Expression, Activation Assay