Journal: Nature Communications

Article Title: CENP-F-dependent DRP1 function regulates APC/C activity during oocyte meiosis I

doi: 10.1038/s41467-022-35461-5

Figure Lengend Snippet: a Co-IP was performed to prove the interaction between CENP-F and DRP1. Per lysate containing 1000 oocytes was incubated with anti-CENP-F and anti-DRP1, respectively. IP eluates were used for immunoblot with anti-CENP-F and anti-DRP1, respectively. The black arrow shows the band of DRP1. b The expression plasmids of PCS2 + - eGFP - Cenp-f C , PCS2 + - eGFP - Cenp-f ΔC/N or PCS2 + - eGFP - Cenp-f ΔN were co-transfected with PCS2 + - cMyc - Drp1 to HEK-293 cells for 48 h, respectively. The lysates were incubated with anti-eGFP. IP eluates were used for immunoblot with anti-eGFP and anti-cMyc, respectively. One-tenth of the input cell lysates were used for immunoblot. c Co-localisation of CENP-F and DRP1 during meiosis I. Enlarged images show representative results. White arrows indicate the measurement direction of fluorescence intensity from the inner to the outer kinetochore. The distance is shown in pixel. Scale bar, 5 μm. d , e Frequency of CENP-F and DRP1 recruited to the kinetochores during meiosis I. White arrows show the measurement direction of the fluorescence intensity. If not defined, the measurement direction starts from the left of the kinetochore to the right in the enlarged images. Fluorescence intensities of CENP-F (green line), DRP1 (red line) and CREST (magenta line) are shown in line graph. The distance shows in pixel. Scale bar, 1 μm. K, kinetochores; C, CENP-F; D, DRP1. f CENP-F and DRP1 localisation on kinetochores after CENP-F or DRP1 Trim-Away. The amplified four images in row 1, row 2 and row 3 show CENP-F and DRP1 co-localised on kinetochores, CENP-F loss reduced DRP1 localisation, and unaffected CENP-F localisation after DRP1 loss, respectively. Scale bar, 5 μm. g Fluorescence intensities of DRP1 and CENPF in ( f ) were measured. The distance is shown in pixel. Chromosomes were stained with Hoechst 33342 (blue), and kinetochores were marked with CREST (magenta in c , d , f ). n in the graphs refers to the number of kinetochores in ( e , g ). The white dotted frames in ( c , d , f ) indicate the region shown in detail. Three independent replicates were performed for ( e , g ). NEBD, nuclear envelope breakdown. Representative blots or stainings from at least three independent repeats are shown. Source data are provided as a Source Data file. See also in Supplementary Figs. – .

Article Snippet: HEK-293 cells (mycoplasma-free) were obtained from American type culture collection (ATCC, CRL-1573) and were cultured in DMEM (GIBCO) supplemented with 10% FBS (ThermoFisher) and 1% penicillin-streptomycin (GIBCO) under the humidified atmosphere of 5% CO 2 at 37 °C.

Techniques: Co-Immunoprecipitation Assay, Incubation, Western Blot, Expressing, Transfection, Fluorescence, Amplification, Staining

Journal: Cell reports

Article Title: Non-acylated Wnts can promote signaling

doi: 10.1016/j.celrep.2018.12.104

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cell culture Mycoplasma-free HEK293STF (ATCC Cat# CRL-3249, RRID:CVCL_AQ26) and Expi293 (RRID:CVCL_D615) cells (human female) were cultured at 37˚C in 5% and 8% CO 2 respectively in a humidified atmosphere.

Techniques: Recombinant, Protease Inhibitor, Luciferase, SYBR Green Assay, Affinity Purification, Expressing, Software

Journal: The Journal of Experimental Medicine

Article Title: Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4

doi: 10.1084/jem.20102660

Figure Lengend Snippet: Gliadin-induced innate immune responses are elicited by wheat ATI, a protein copurifying with ω-gliadins. (A) Stimulation of THP-1 cells with α-, γ-, ω1.2-, and ω5-gliadin fractions (all 100 µg/ml) isolated from the pure wheat strain Rektor. Co-incubation of α- and γ-gliadin with 100 µg/ml of regular PT gliadin from Sigma-Aldrich served as cell viability control. LPS was used as positive control, whereas PT or PT zein served as negative control. (B and C) IL-8 secretion after stimulation with 100 µg/ml ω-gliadins in TLR4-transfected (B) and in untransfected HEK-293 cells (C). 10 ng/ml PMA served as cell viability control. 10 ng/ml LPS, 100 µg/ml PT gliadin, or 100 µg/ml of a PT digest of Rektor gliadin (PT Rektor) served as positive control, and 100 µg/ml PT zein, 1 µg/ml Pam3CSK4, or a PT mixture (PT ctrl) served as negative controls. (D) Stimulatory capacity of synthetic overlapping 20mers of ω5-gliadin in TLR4-transfected HEK-293 cells. For illustration purposes, 9 fractions each were pooled in the stimulation experiments (each fraction at a concentration of 100 µg/ml), while also all 43 fractions were tested individually. LPS served as positive and Pam3CSK4 or PT zein as negative controls. (E and F) Dose response of IL-8 release by monocyte-derived DCs stimulated with water-soluble (ws) gliadin (which is enriched in ATI; E) or with purified ATI (F). LPS and water-soluble zein served as positive and negative controls, respectively. (G) Secretion of IL-12 in monocyte-derived DCs from healthy subjects upon stimulation with ATI and PT gliadin in the presence of 1,000 U/ml Interferon-γ as co-stimulatory protein. LPS and PT zein served as positive and negative controls, respectively. (H) Effect of proteinase K digestion of ATI and LPS on IL-8 secretion in DCs. (I) KC secretion in peritoneal macrophages isolated from MyD88 −/− mice compared with C57BL/6J wild-type mice upon ATI or water-soluble gliadin stimulation. LPS and water-soluble zein served as positive and negative controls, respectively. (J) IL-8 secretion of monocyte-derived DCs stimulated with ATI and LPS after preincubation with anti-TLR4 or anti-CD14 antibodies. TLR2 agonist Pam3CSK4 served as positive control. *, P < 0.05 versus negative (positive) control (if not indicated otherwise); all graphs illustrate representative data from one of at least three independent experiments, all performed in triplicates. Error bars depict standard errors of the mean.

Article Snippet: To exclude bacterial contaminants, recombinant flag-tagged ATI CM3 and 0.19 were generated in eukaryotic mycoplasma-free HEK-293 cells, using cDNAs optimized to fit eukaryotic codon usage (Genscript; CM3 and 0.19 with GenBank accession numbers AY436554.1 and AY729672.1 , respectively).

Techniques: Isolation, Incubation, Positive Control, Negative Control, Transfection, Concentration Assay, Derivative Assay, Purification

Journal: The Journal of Experimental Medicine

Article Title: Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4

doi: 10.1084/jem.20102660

Figure Lengend Snippet: Identification of ATI CM3 and 0.19 as inducers of innate immune responses. (A) Coimmunoprecipitation of a soluble flag-tagged TLR4/MD2 fusion protein with or without biotinylated ATI. Pull-down was performed by streptavidin-agarose. Western blot (WB) analysis using a rabbit anti-flag antibody was performed to detect the TLR4/MD2 fusion protein. The image illustrates representative data from one of three independent experiments. (B) CM3 and 0.19 were expressed in HEK-293 cells and purified as Flag-tagged molecules, yielding expected molecular masses of 15 kD and 17 kD, respectively, after 15% SDS-PAGE and Western blotting with Flag antibody. There was no expression in mock (vector only)-transfected cells. 0.19 was not secreted into media but could be recovered in SDS/DTT buffer (Cell), deoxycholate and Triton X-100 buffer (Sup), or the remaining cell pellet. (C) Purity of CM3 and 0.19 ATI after affinity purification on Flag-agarose (15% SDS-PAGE) after staining with Coomassie blue or blotting with Flag antibody. (B and C) Arrows denote expected molecular masses of CM3 and 0.19 ATIs. (D) IL-8 secretion by TLR4–CD14–MD2-expressing HEK-293 cells after stimulation with recombinant ATI. Cells were left untreated or stimulated with affinity-purified detergent extracts from mock-transfected cells, from recombinant CM3, 0.19, or with ATI isolated from wheat (all 5 µg/ml). Controls were 5 µg/ml Flag peptide, 100 µg/ml PT gliadin, and 10 ng/ml LPS. (E) Effect of expression of CM3 and 0.19 in HEK-TLR4 cells on downstream signaling of the canonical (pNF-κB/p65) and the alternative (pIRF3) pathways. Western blots of whole cell lysates (50 µg protein). (F) Reduction and alkylation (R/A) of enriched ATIs from spring wheat (ABic: ammonium bicarbonate extract; AA: acetic acid extract). Stimulatory capacity was measured by IL-8 secretion in U-937 cells. LPS served as positive control. *, P < 0.05 versus negative (positive) control. All graphs illustrate representative data from one of at least three independent experiments. D and F were performed in triplicates. Error bars depict standard errors of the mean.

Article Snippet: To exclude bacterial contaminants, recombinant flag-tagged ATI CM3 and 0.19 were generated in eukaryotic mycoplasma-free HEK-293 cells, using cDNAs optimized to fit eukaryotic codon usage (Genscript; CM3 and 0.19 with GenBank accession numbers AY436554.1 and AY729672.1 , respectively).

Techniques: Western Blot, Purification, SDS Page, Expressing, Plasmid Preparation, Transfection, Affinity Purification, Staining, Recombinant, Isolation, Positive Control