Journal: International journal of biological macromolecules

Article Title: Disulfide bond and crosslinking analyses reveal inter-domain interactions that contribute to the rigidity of placental malaria VAR2CSA structure and formation of CSA binding channel.

doi: 10.1016/j.ijbiomac.2022.11.258

Figure Lengend Snippet: Fig. 8. The binding capacity of full-length VAR2CSA ectodomain is lower than the ectodomain lacking NTS. The binding of NTS-DBL6ε and DBL1x-DBL6ε to CSPG was measured in triplicates by ELISA. The wells of 96-well microtiter plates were coated with CSPG (200 ng/ml) and blocked with BSA and then incubated with 1:2 serially diluted solutions of NTS-DBL6ε or DBL1x-DBL6ε. The levels of bound proteins were measured using anti-cMyc antibody and HRP- conjugated secondary antibody. Data are a representative of three independent experiments using different batches of purified proteins. KD values were determined by plotting OD vs protein concentration using GraphPad Prism v9.4.1. KD ± SE: 41.5 ± 3.9 (DBBL1x-DBL6ε); 59.9 ± 6.8 (NTS-DBL6ε).

Article Snippet: Unbound proteins in the coated wells were removed by washing with 100 μl of PBST and the plates were incubated with 1:1000 diluted mouse anti-cMyc monoclonal antibody (Cat No. NB600-302, NOVUS Biologicals, Centennial, CO), and washed three times with 100 μl PBST.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Purification, Protein Concentration

Journal: The Journal of Biological Chemistry

Article Title: c-Myc is a novel Leishmania virulence factor by proxy that targets the host miRNA system and is essential for survival in human macrophages

doi: 10.1074/jbc.RA118.002462

Figure Lengend Snippet: c-Myc knockdown and inhibition reverse miRNA down-regulation and Drosha up-regulation. A, HMDMs were treated for 48 h with control (Scrambled) and c-Myc siRNAs (siRNA A, B, and C), followed by infection for 24 h, lysis, and analysis by Western blotting for c-Myc and actin. Densitometry analysis results are shown below (means ± S.D., n = 3 donors; two-tailed t test). B, RNA was collected from siRNA-treated cells (siRNA B and C), both infected (Ld) and control, and RT–qPCR was performed to assess the expression of U6 and six miRNAs. Levels of miRNA expression are depicted as log2 fold change (FC) compared with scrambled and normalized to U6 (means ± S.D., n = 3 donors; two-way ANOVA comparing each condition). C, HMDMs were treated with c-Myc inhibitor 10058-F4 (F4, 25 or 50 μm) or DMSO for 24 h and then infected for 24 h. Drosha expression was assessed by Western blotting (means ± S.D., n = 3 donors; two-tailed t test). D, RNA was extracted from 10058-F4–treated cells, both infected (Ld) and control cells, and analyzed by RT–qPCR. The expression levels of six miRNAs are depicted as log2 fold change compared with DMSO and normalized to U6 (means ± S.D., n = 4 donors; two-way ANOVA). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: ImageJ was used to determine the fluorescence intensity in the nucleus versus the cytoplasm: DAPI-positive regions were selected as “nucleus,” and the intensity of the DGCR8 or Drosha staining was measured in the DAPI regions, whereas the staining in the rest of the image was measured for cytoplasmic localization. siRNA transfection and 10058-F4 treatment The siRNAs—three c-Myc siRNAs duplexes and one scrambled negative control—were obtained from ORIGENE (SR321047).

Techniques: Knockdown, Inhibition, Control, Infection, Lysis, Western Blot, Two Tailed Test, Quantitative RT-PCR, Expressing

Journal: The Journal of Biological Chemistry

Article Title: c-Myc is a novel Leishmania virulence factor by proxy that targets the host miRNA system and is essential for survival in human macrophages

doi: 10.1074/jbc.RA118.002462

Figure Lengend Snippet: c-Myc knockdown and inhibition attenuate Leishmania survival. A, HMDMs were treated for 48 h with control (Scrambled) and c-Myc siRNAs (siRNA B and C), followed by infection for 24 h. A parasite rescue assay as described under “Experimental procedures” was performed (means ± S.D., n = 3 donors; two-tailed t test). B, HMDMs were treated with c-Myc inhibitor 10058-F4 (F4, 25 or 50 μm) or DMSO for 24 h and then infected for 24 h. A parasite rescue assay was performed (means ± S.D., n = 4 donors; two-tailed t test). *, p < 0.05; **, p < 0.01.

Article Snippet: ImageJ was used to determine the fluorescence intensity in the nucleus versus the cytoplasm: DAPI-positive regions were selected as “nucleus,” and the intensity of the DGCR8 or Drosha staining was measured in the DAPI regions, whereas the staining in the rest of the image was measured for cytoplasmic localization. siRNA transfection and 10058-F4 treatment The siRNAs—three c-Myc siRNAs duplexes and one scrambled negative control—were obtained from ORIGENE (SR321047).

Techniques: Knockdown, Inhibition, Control, Infection, Rescue Assay, Two Tailed Test