Journal: bioRxiv

Article Title: Localization of planteose hydrolysis during seed germination of Orobanche minor

doi: 10.1101/2021.06.16.448768

Figure Lengend Snippet: Localization of mCherry fusion proteins transiently expressed in leaves of Nicotiana benthamiana . (A) OmAGAL2:mCherry, (E) ΔSP-OmAGAL2:mCherry, (I) SP:mCherry, and (M) mCherey were co-expressed with the apoplast marker At5g11420:pH-tdGFP (B, F, J, N). (C, D, G, H, K, L, O, P) Merged images of fluorescence from mCherry and GFP. Scale bars: 50 µm (A–C, E–G, I–K, M–O) and 20 µm (D, H, L, P).

Article Snippet: Transient expression was conducted by co-inoculation of Agrobacterium tumefaciens strain GV3101 cultures carrying each construct with those carrying the vector pMDC-At5g11420:pH-tdGFP as an apoplast marker , and pDGB3alph2_35S:P19:Tnos (GB1203, Addgene #68214) in leaves of Nicotiana benthamiana . mCherry and green fluorescent protein (GFP) were excited at 555 and 488 nm, and observed in the range of 570–600 and 490–520 nm, respectively, using a LSM700 laser scanning confocal microscope (Carl Zeiss, Jena, Germany).

Techniques: Marker, Fluorescence

Journal: PLOS Pathogens

Article Title: HBZ upregulates myoferlin expression to facilitate HTLV-1 infection

doi: 10.1371/journal.ppat.1011202

Figure Lengend Snippet: (A) Inhibition of MyoF reduces levels of SU (gp46). ATL-2 cells were treated with DMSO or WJ460 (200 nM or 1 μM) for 24h. Whole cell extracts (50 μg) from uninfected CEM and the treated cells were analyzed by Western blot using antibodies against MyoF, β-actin and the viral proteins Tax, Gag p55, Gag p19, and SU/Pr. (B) ATL-2 and SLB-1 cells were treated with DMSO or 1 μM WJ460 for 24h. Whole cell extracts (100 μg for Tax; 50 μg for the others) were analyzed by Western blot using antibodies against β-actin and the viral proteins Tax, and SU/Pr. (C) The graph shows quantification of band intensities of Pr (gp62) and SU (gp46) normalized to band intensities of β-actin averaged from three and four independent experiments for ATL-2 and SLB-1 cells, respectively. Error bars show standard deviations; * p <0.05, ** p <0.01. (D) Knockdown of MyoF expression reduces levels of SU (gp46). Whole cell extracts were prepared from SLB-1 cells stably expressing an shRNA targeting GFP (negative control) or MYOF mRNA, and 50 μg from each cell line was analyzed by Western blot using antibodies against MyoF, β-actin and the viral proteins Tax, Gag p19, and SU/Pr. (E) The graph shows quantification of band intensities of Pr (gp62) and SU (gp46) normalized to band intensities of β-actin averaged from three independent experiments. Error bars show standard deviations; ** p <0.01. (F) Ectopic co-expression of MyoF with Env in HEK293T cells increases SU (gp46) abundance. Cells (1 x 10 6 ) were transfected with pcDNA-GFP-HA-MyoF (3 μg) and pCMV-HTLV-1-Env (1 μg). Env was co-precipitated with Lens Culinaris Agglutinin lectin bound to agarose beads (P-lectin) from 879 μg of whole cell extracts (WCE) and analyzed along with 50 μg WCE by Western blot using antibodies against MyoF, and SU/Pr. (G) Inhibition of MyoF ectopically co-expressed with Env in HEK293T cells decreases SU/gp46 abundance. Cells (1 x 10 6 ) were transfected with pcDNA-GFP-HA-MyoF (3 μg) and pCMV-HTLV-1-Env-Flag-Myc (3 μg) expression vectors and treated with DMSO or WJ460 (1 μM) for 24h. Env was co-precipitated with Lens Culinaris Agglutinin lectin bound to agarose beads (P-lectin) from 768 μg of whole cell extracts (WCE) and analyzed along with 50 μg WCE by Western blot using antibodies against MyoF, β-actin and the Flag-epitope tag of Env. (H) Ectopic co-expression of MyoF with Env in HEK293T cells increases TM (gp21) abundance. Cells (1 x 10 6 ) were transfected with pcDNA-GFP-HA-MyoF (3 μg) and pCMV-HTLV-1-Env-Flag-Myc (3 μg). Env was co-precipitated with Lens Culinaris Agglutinin lectin bound to agarose beads (P-lectin) from 1190 μg of whole cell extracts (WCE) and analyzed along with 50 μg WCE by Western blot using antibodies against MyoF, and the Flag- and Myc-epitope tags of Env.

Article Snippet: Antibodies used were as follows: anti-HTLV-1 Env (ARP-1578) and anti-Tax (hybridoma 168B17-46-92) were obtained from NIH AIDS Research and Reagent Program; anti-HBZ has been described [ ]; anti-6x His (ab9108) was purchased from Abcam; anti-MyoF (HPA014245) and anti-Myc (06–340) were purchased from MilliporeSigma; anti-HTLV-1 Env gp46 (1C11, sc-53890; 65/6C2.2.34, sc-57865), anti-Gag p19 (TP7, sc-57870) and anti-β-actin (C4, sc-47778) were purchased from Santa-Cruz; anti-clathrin (D3C6, #4796), anti-caveolin-1 (D46G3, #3267), anti-EEA1 (C45B10, #3288), anti-Rab5a (E6N8S, #46449), anti-Rab7 (D95F2, #9367), anti-Rab11 (D4F5, #5589), anti-LAMP-1 (D2D11, #9091) were purchased from Cell Signaling; anti-EHD2 (PA5-80576) was purchased from ThermoFisher Scientific.

Techniques: Inhibition, Western Blot, Knockdown, Expressing, Stable Transfection, shRNA, Negative Control, Transfection, FLAG-tag

Journal: PLOS Pathogens

Article Title: HBZ upregulates myoferlin expression to facilitate HTLV-1 infection

doi: 10.1371/journal.ppat.1011202

Figure Lengend Snippet: (A) Knockdown of MyoF expression reduces overall levels of SU in the culture medium of HTLV-1 cells. TCA-precipitated proteins from culture media of SLB-1 cells stably transfected with shRNA targeting GFP or MYOF mRNA were divided into equal fractions and analyzed by Western blot using an antibody against SU/Pr (upper panel) and, separately, by Coomassie blue-staining following SDS-PAGE (lower panel). Whole cell extract from SLB-1 cells served as a positive control. (B) Knockdown of MyoF expression reduces levels of SU in cell-free HTLV-1 virions. Culture media from SLB-1 cells stably expressing an shRNA targeting GFP or MYOF mRNA were filtered, ultracentrifuged and analyzed by Western blot using antibodies against SU/gp46 and Gag p19; * denotes a nonspecific band from the serum. (C) Knockdown or inhibition of MyoF does not affect levels of virus released from the cells. Clarified culture media from SLB-1 cells stably expressing an shRNA targeting GFP or MYOF mRNA and SLB-1 and ATL-2 cells treated with DMSO or 1 μM WJ460 for 24 h were analyzed by ELISA to quantify levels of Gag p19. The graph shows values averaged from three replicates.

Article Snippet: Antibodies used were as follows: anti-HTLV-1 Env (ARP-1578) and anti-Tax (hybridoma 168B17-46-92) were obtained from NIH AIDS Research and Reagent Program; anti-HBZ has been described [ ]; anti-6x His (ab9108) was purchased from Abcam; anti-MyoF (HPA014245) and anti-Myc (06–340) were purchased from MilliporeSigma; anti-HTLV-1 Env gp46 (1C11, sc-53890; 65/6C2.2.34, sc-57865), anti-Gag p19 (TP7, sc-57870) and anti-β-actin (C4, sc-47778) were purchased from Santa-Cruz; anti-clathrin (D3C6, #4796), anti-caveolin-1 (D46G3, #3267), anti-EEA1 (C45B10, #3288), anti-Rab5a (E6N8S, #46449), anti-Rab7 (D95F2, #9367), anti-Rab11 (D4F5, #5589), anti-LAMP-1 (D2D11, #9091) were purchased from Cell Signaling; anti-EHD2 (PA5-80576) was purchased from ThermoFisher Scientific.

Techniques: Knockdown, Expressing, Stable Transfection, Transfection, shRNA, Western Blot, Staining, SDS Page, Positive Control, Inhibition, Virus, Enzyme-linked Immunosorbent Assay