Journal: Science Advances

Article Title: Norovirus co-opts NINJ1 for selective protein secretion

doi: 10.1126/sciadv.adu7985

Figure Lengend Snippet: ( A ) WT or Ninj1 KO BV2 cells were infected with CR6 at 5 MOI and harvested at 10 hpi. Cell lysates were immunoprecipitated with anti-NS1 and analyzed by immunoblotting. ( B and C ) Transmission electron microscopy image of uninfected (B) or CR6-infected (C) BV2 cells with immunogold labeling for FLAG-NINJ1 (12-nm gold, black arrowheads) and NS1 (18-nm gold, red arrowheads). RC, replication complex. Scale bar, 500 nm. ( D ) Representative immunofluorescence confocal imaging of FLAG-NINJ1 (red), NS1 (green), NS6/7 (cyan), and 4′,6-diamidino-2-phenylindole (DAPI, blue) in Ninj1 KO BV2 cells reconstituted with FLAG-NINJ1 WT or K45Q at 14 hpi. White arrowhead highlights the colocalization of NINJ1 and NS1 proteins. Scale bars, 5 μm. All images are representative of three or more independent experiments. ( E ) Quantification of colocalization between NS1 and FLAG-NINJ1 WT or K45Q. Bars represent means ± SEM, n = 10 to 11 cells. ( F ) Dose-dependent binding of Fc-tagged ectodomain of human NINJ1 (hNINJ1) or mouse NINJ1 (mNINJ1) protein to recombinant NS1 protein was determined by ELISA. A monoclonal anti-NS1 (clone CM79) and isotype control were included as a positive and negative control, respectively. Data represent means ± SEM of duplicates, and the dissociation constants ( K d ) are shown. ( G to I ) Direct binding between recombinant NS1 and mNINJ1 protein was measured by MST. ΔFnorm indicates the change in thermophoresis, and K d is shown. n.d., not determined. (G) Dose-response curve showing bindings of WT NS1 protein to fluorescence-labeled WT or mutated mNINJ1 proteins. (H) Dose-response curve showing bindings of WT or mutated NS1 proteins to fluorescence-labeled mNINJ1. Maltose-binding protein (MBP) was included as a negative control. (I) Dose-response curve showing bindings of WT or mutated NS1 proteins to fluorescence-labeled monoclonal anti-NS1 or isotype control. Data are shown in means ± SEM ( n = 3).

Article Snippet: Primary antibodies used in this study include the following: Strep-tag II (A01732, Genscript), FLAG (for immunoblotting; 200-350-383, Rockland Immunochemicals), FLAG (for immunofluorescence; 637303, BioLegend), 6xHis (ab18184, Abcam), GSDMD (ab209845, Abcam), active caspase-3 (clone C92-605; 559565, BD Biosciences), glyceraldehyde-3-phosphate dehydrogenase (MCA4739, Bio-Rad), calnexin (ab22595, Abcam), RAB7 (ab137029, Abcam), ATP5A1 (14676-1-AP, Proteintech), and TGN46 (ab16059, Abcam).

Techniques: Infection, Immunoprecipitation, Western Blot, Transmission Assay, Electron Microscopy, Labeling, Immunofluorescence, Imaging, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Control, Negative Control, Fluorescence

Journal: Frontiers in Plant Science

Article Title: DeepCount : In-Field Automatic Quantification of Wheat Spikes Using Simple Linear Iterative Clustering and Deep Convolutional Neural Networks

doi: 10.3389/fpls.2019.01176

Figure Lengend Snippet: Characteristics of the three experiments considered in this study.

Article Snippet: 2016-WGIN , 360 , 0, 100, 200, 350 , 78 , Canon G12 , 3,648 × 2,736 , 6 mm , 0.21–0.31 , 13/06/2016.

Techniques: