Journal: bioRxiv

Article Title: Actomyosin contractility modulates Wnt signaling through adherens junction stability

doi: 10.1101/220178

Figure Lengend Snippet: (A,B) Wnt-responsive luciferase TOPFLASH assay measuring TCF/LEF reporter activity in (A) MCF7 and (B) RKO cells following Wnt3A transfection and siPP1β and siMYPT3 transfection. Data are presented as mean ± SEM with letters above representing significant difference from corresponding column, (P<0.01). (C) Western blot analysis of total cellular levels of E-cad, β-cat, MYPT3, Wnt3A and PP1β. β-tub was used as a loading control. (D) Western blot analysis of β-cat in cytoplasmic (Cyto), membranous (Mem), chromatin-bound (CB), and whole cell extract (WCE) fractions in MCF7 cells. GAPDH, Na + /K + ATPase, and Histone 3 were used as loading controls for corresponding fractions. Complete fraction, see . (E) Quantification of relative levels of β-cat from each fraction taken from (D), normalized to Control + siScramble conditions. Data shown as mean ± SEM (n = 4 biological replicates), ns = not significant, *P<0.05.

Article Snippet: Proteins were transferred onto nitrocellulose membranes, and probed against the following primary antibodies: rabbit anti-β-catenin (1:1000, Cell Signaling), rabbit anti-E-cadherin (1:1000, Cell Signaling), mouse anti-PPP1R16A (MYPT-3) (1:500, abcam), mouse anti-Protein Phosphatase 1 beta (PP1β) (1:1000, abcam), rabbit anti-Wnt3A (1:1000, Cell Signaling), mouse anti-β-tubulin (1:1000, ABM), rabbit anti-GAPDH (1:3000, Cell Signaling), mouse anti-Na+/K+ ATPase (1:50 DSHB), rabbit anti-Histone H3 (1:1000 Cell Signaling).

Techniques: Luciferase, TOPFlash assay, Activity Assay, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: Active EB1 surges promote tubulin influx into the growing outer segments of the bipartite olfactory cilia in Drosophila

doi: 10.1101/2024.09.10.612170

Figure Lengend Snippet: A) Copurification of EB1:GFP from the chaGal4>EB1:GFP expressing Drosophila head extracts with the recombinant Glutathione S-transferase (GST)-tagged, KLP68D tail (GST-KLP68DT) fragment using affinity chromatography. The arrow indicates the EB1:GFP band. B) The immune-coprecipitation (IP) of the recombinant KLP68D from the head extracts of chaGal4>Klp68D:YFP and chaGal4>Klp68D(ΔT)YFP, expressing UAS-EB1FLAG using anti-FLAG. The arrow indicates a full-length KLP68DYFP band.

Article Snippet: Proteins from these gels were transferred onto a previously activated PVDF membrane (Hybond-P, GE Healthcare Ltd) in an electro-blotting apparatus (Bio-Rad, USA) following the supplier’s protocol and incubated in different primary antisera solutions as the following: anti-GFP Rabbit (dilution, 1:500; #3999 Bio Vision Inc., CA, USA) or anti-FLAG Rabbit (1:500; #F7425 Sigma-Aldrich, USA) Or anti-GST mouse (1:1000, Bioklone Biotech Pvt.

Techniques: Copurification, Expressing, Recombinant, Affinity Chromatography

Journal: bioRxiv

Article Title: Actomyosin contractility modulates Wnt signaling through adherens junction stability

doi: 10.1101/220178

Figure Lengend Snippet: (A) Salivary glands from control or dpp>flw-RNAi stained for fz3 expression. (B) Localization of Dsh-GFP and FLAG-Axin in the proximal cells of the salivary gland, identified in the dashed line area of (A), (C-C”) Effects of flw-RNAi on ectopic Dll-lacZ in wing imaginal discs: (C) RFP marked flip-out clones expressing Fz-Arr and GFP or flw-RNAi (C) GFP-positive axin nul1 MARCM clones and flw-RNAi in axin nul1 MARCM clones. (C”) Arm S10 flip out clones with GFP or flw-RNAi. (D,D’) Effects of flw-RNAi on Arm distribution in GFP-marked axin null cells. (D) DAPI was used to identify nuclei, and F-actin to mark the edges of the cell. (D’) Percent of nuclear Arm in cells was measured as an intensity plot (dotted line D) in wild type (n = 16), axin nul1 (n = 20), and axin null , flw-RNAi cells (n = 15). Data presented as mean ± SEM; ***p< 0.001. Scale bars: (A) 100 μm, (B-C”) 50 μm, (D) 5 μm.

Article Snippet: The following primary antibodies and dilutions were used: mouse anti-β-galactosidase (1:2000 Promega), mouse anti-Wg (1:100 DSHB), mouse anti-Arm (1:50 DSHB), rabbit anti-cleaved Caspase 3 (1:100 Cell Signaling), guinea pig anti-Sens (1:500, a gift from Hugo Bellen, Dept. of Molecular and Human Genetics, Baylor College of Medicine, USA), mouse anti-Dll (1:300, a gift from Ian Duncan, Dept. of Biology, Washington University in St. Louis, USA), rabbit anti-Phospho-Myosin Light Chain 2 (Ser19) (p-MyoII) (1:25 Cell Signaling), mouse anti-GFP (1:500, Cell Signaling), rabbit anti-FLAG (1:200, Sigma), rat anti-DEcad (extracellular domain) (1:50 DSHB), rat anti-Ci (1:50, DSHB).

Techniques: Staining, Expressing, Clone Assay

Journal: bioRxiv

Article Title: N-Cadherin Provides a Cis and Trans Ligand for Astrotactin that Functions in Glial-Guided Neuronal Migration

doi: 10.1101/357541

Figure Lengend Snippet: Flow cytometry of live HEK 293T cells labeled with GFP and Alexa Fluor 647 antibodies. Control, untransfected cells (A) or cells transfected with Venus (B) , Astnl-FL-Venus (C) , or Astnl-ΔCTD-Venus , lacking the MACPF, FNIII and ANX-like domains in the C-terminus (D) . The x-axis shows total GFP fluorescence and the y-axis shows surface labeling (Alexa Fluor 647). Thus, cells expressing cytosolic Venus-tagged proteins are indicated in the lower right quadrant (Q3), while double positive cells (GFP+/Alexa Fluor 647+) in the upper right quadrant (Q2) express Venus-tagged proteins exposed on the cell surface. The percentage of cells expressing ASTN1 on the cell surface is depicted. Both ASTN1-FL and ASTNi-ΔCTD were significantly expressed on the cell surface (58 % and 49 % of live transfected cells, respectively).

Article Snippet: After removing the beads, the lysates were incubated with 3 μg of a rabbit GFP antibody (Invitrogen), rabbit Astn1 antibody, or normal rabbit IgG (Santa Cruz Biotechnology) for 2 h at 4 o C. Immunoprecipitates were collected on 50 μl Protein G/A Agarose beads by overnight rotation at 4 o C, washed with lysis buffer and resuspended in 50 μl 2X Laemmli buffer.

Techniques: Flow Cytometry, Labeling, Transfection, Fluorescence, Expressing

Journal: bioRxiv

Article Title: N-Cadherin Provides a Cis and Trans Ligand for Astrotactin that Functions in Glial-Guided Neuronal Migration

doi: 10.1101/357541

Figure Lengend Snippet: Drosophila S2 cell adhesion assays were prepared in four conditions: Cdh2;GFP + mCherry;GFP (A) , Cdh2;GFP + Astn1;mCherry (B) , Cdh2;GFP + Astn1;mCherry + ASTN1 Fab (C) , and Cdh2-A390;GFP + Astn1;mCherry (D) . ASTN1-positive cells were adhering to the CDH2-expressing aggregates after 30 min (arrows in B1 ), with more co-aggregation seen after 1 h (arrows in B2 ) and 2 h (arrows in B3 ), indicating heterophilic trans interactions. Significantly lower proportions of cells were adhering to the aggregates in the conditions with cells expressing control vector (A) or Astn1;mCherry blocked with ASTN1 Fab fragments (C) . The proportion of mCherry expressing cells in the CDH2;GFP-positive aggregates is quantified in (E) . Expression of CDH2∆390;GFP did not result in cell aggregation within 2 h (D) , demonstrating the importance of the cadherin ectodomain for homophilic and heterophilic interactions and cell adhesion. ** P < 0.01; *** P < 0.001. Scale bar represents 20 μm in (A - D) and 10 μm in inset in (B3).

Article Snippet: After removing the beads, the lysates were incubated with 3 μg of a rabbit GFP antibody (Invitrogen), rabbit Astn1 antibody, or normal rabbit IgG (Santa Cruz Biotechnology) for 2 h at 4 o C. Immunoprecipitates were collected on 50 μl Protein G/A Agarose beads by overnight rotation at 4 o C, washed with lysis buffer and resuspended in 50 μl 2X Laemmli buffer.

Techniques: Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: SORLA-driven endosomal trafficking regulates the oncogenic fitness of HER2

doi: 10.1101/299586

Figure Lengend Snippet: ( a ) Western blot analysis of SORLA and HER2 in bladder cancer cell lines (5637 and T24) and three patient-derived cell cultures. α-tubulin is a loading control. ( b ) Confocal microscopy imaging of endogenous SORLA or SORLA-GFP (green) with endogenous EEA1, VPS35, LAMP1, Rab11, Rab5 (magenta) or Rab7-mRFP (magenta; Rab7 was overexpressed due to lack of reliable Rab7 antibodies for staining) in MDA-MB-361 (top panel) or JIMT-1 cells. ( c ) Endogenous HER2 (magenta) and EEA1 (green) staining in SKBR3, HCC1419 and HCC1954 cells. ( d ) Co-localization between SORLA-GFP and HER2 in JIMT-1 cells (n = 130 cells from 7 independent experiments). Details of colocalization analysis can be found in the methods section. ( e ) Live-cell confocal imaging of AlexaFluor-568-labeled trastuzumab (Tz-568; red) and SORLA-GFP (green) in the cytoplasm of MDA-MB-361 cells, 250 ms frames. ( f ) Confocal imaging of different SORLA-GFP fusion proteins in MDA-MB-361 cells. ECD: extracellular domain; TM: transmembrane domain; CD: cytosolic domain.

Article Snippet: Lab Vision autostainer (Thermo Fisher Scientific) with SORLA antibody (rabbit polyclonal, Atlas Antibodies; 1:300 dilution) was used and primary antibodies were detected with PowerVision Poly-HRP anti-mouse/anti-rabbit IHC system (Leica BioSystems).

Techniques: Western Blot, Derivative Assay, Confocal Microscopy, Imaging, Staining, Labeling

Journal: bioRxiv

Article Title: E2F3-dependent activation of FAM111B restricts mouse cytomegalovirus replication in primate cells

doi: 10.1101/2024.08.02.606359

Figure Lengend Snippet: (A) RPE-1 cells were arrested in G0 by contact inhibition and 48 h serum starvation (-FCS 48 h), in G1 by 24 h serum starvation (-FCS 24 h), in S phase by 24 h treatment with 1 mM hydroxyurea (HU) and in G2/M by 24 h treatment with 200 ng/ml nocodazole. Asynchronous (asynchr.) cells are shown for comparison. The cells were fixed and stained with propidium iodide. The DNA content was analyzed by flow cytometry to verify synchronization. Cells in G0 and G1 have a DNA content of 2n, cells in S phase between 2n and 4n, and cells in G2/M a 4n DNA content. The percentages of cells in each cell cycle phase are indicated. (B) FAM111A and FAM111B expression in cell-cycle arrested cells was analyzed by immunoblot. Cyclin E and cyclin B1 were analyzed as markers for S and G2/M phases, respectively. GAPDH was used as a loading control. ( C) Subconfluent RPE-1 cells were treated with an E2F inhibitor (HLM0006474, 40 μM) or vehicle (DMSO). Cell lysates were harvested at the indicated times (hours post-treatment, hpt) and analyzed by immunoblot. (D to F) WT RPE-1 cell, E2F1 KO, and E2F3 KO cells were synchronized by serum starvation for 24 h and infected with MCMVs expressing full-length or mutant M117 (MOI 5). Cell lysates were harvested at the indicated times post-infection and analyzed by immunoblot. The MCMV immediate-early 1 (IE1) protein and β-actin were used as infection and loading controls, respectively. Representative blots out of multiple experiments are shown. hpt: hours post treatment.

Article Snippet: Polyclonal rabbit antibodies against FAM111B (HPA038637, Sigma-Aldrich), FAM111A (EPR14407, Abcam), Flag (F7425, Sigma-Aldrich), E2F3 (C-18, Santa Cruz) and E2F4 (C-20, Santa Cruz) were used.

Techniques: Inhibition, Comparison, Staining, Flow Cytometry, Expressing, Western Blot, Control, Infection, Mutagenesis

Journal: bioRxiv

Article Title: E2F3-dependent activation of FAM111B restricts mouse cytomegalovirus replication in primate cells

doi: 10.1101/2024.08.02.606359

Figure Lengend Snippet: (A) RPE-1 cells were infected (MOI 5 TCID 50 /cell) with recombinant MCMV-GFP encoding FAM111A-specific (A sh1, A sh2), FAM111B-specific (B sh1, B sh2), or scrambled (scr) control shRNA. Cell lysates were harvested 24 and 28 hpi and analyzed by immunoblot. (B) RPE-1 cells (MOI 0.2), (C) ARPE-19 (MOI 1) and (D) RPTEC (MOI 1) were infected with the same viruses as above. Viral replication kinetics were determined by titration of virus released into the supernatant. (C) Rhesus fibroblasts were infected (MOI 1.5 TCID 50 /cell) with the same viruses as above and analyzed by immunoblot or infected at MOI 1 TCID 50 /cell (D) with the same viruses as above. Viral replication kinetics were determined by titration of virus released into the supernatant. Mean ±SEM of triplicates are shown. DL, detection limit. (E, F) Cell lysates of WT RPE-1 cells, empty vector (EV)-transduced RPE-1 cells, two FAM111B KO RPE-1 clones (cl 1-9 and 3-18), and one FAM111A KO RPE-1 (cl 3-10) clone were analyzed by immunoblot. (G) Multistep replication kinetic of WT MCMV in the same RPE-1 as in E. Supernatants of infected cells were harvested at the indicated times post infection and titrated. Mean ±SEM of triplicates are shown. DL, detection limit.

Article Snippet: Polyclonal rabbit antibodies against FAM111B (HPA038637, Sigma-Aldrich), FAM111A (EPR14407, Abcam), Flag (F7425, Sigma-Aldrich), E2F3 (C-18, Santa Cruz) and E2F4 (C-20, Santa Cruz) were used.

Techniques: Infection, Recombinant, Control, shRNA, Western Blot, Titration, Virus, Plasmid Preparation, Clone Assay

Journal: bioRxiv

Article Title: E2F3-dependent activation of FAM111B restricts mouse cytomegalovirus replication in primate cells

doi: 10.1101/2024.08.02.606359

Figure Lengend Snippet: (A) 10.1 mouse fibroblasts were infected (MOI 1 TCID 50 /cell) with recombinant MCMVs encoding HA-tagged WT or mutant FAM111B. Cell lysates were analyzed by immunoblot. (B) 10.1 cells were infected (MOI 0.02) with the same viruses as above to analyze multistep replication kinetics. Mean ±SEM of triplicates are shown. DL, detection limit. (C) NIH-3T3 fibroblasts transduced with a lentiviral vector encoding tet-inducible FAM111B were induced with 2 µg/ml doxycyline or left untreated. Cell lysates were harvested and analyzed by immunoblot. (D) Transduced NIH-3T3 cells were treated with doxycycline 1h before (−1 hpi) or 4 h after (+4 hpi) infection with WT MCMV (MOI 0.02) to analyze multistep replication kinetics. Mean ±SEM of triplicates are shown.

Article Snippet: Polyclonal rabbit antibodies against FAM111B (HPA038637, Sigma-Aldrich), FAM111A (EPR14407, Abcam), Flag (F7425, Sigma-Aldrich), E2F3 (C-18, Santa Cruz) and E2F4 (C-20, Santa Cruz) were used.

Techniques: Infection, Recombinant, Mutagenesis, Western Blot, Transduction, Plasmid Preparation

Journal: bioRxiv

Article Title: E2F3-dependent activation of FAM111B restricts mouse cytomegalovirus replication in primate cells

doi: 10.1101/2024.08.02.606359

Figure Lengend Snippet: (A) Immunofluorescence of 10.1 fibroblasts mock-infected or infected with MCMV-eGFP-FAM111B (MOI 1 TCID 50 /cell). Cells were fixed 24 hpi and stained with an antibody recognizing the MCMV E1 protein (red), a marker for vRCs. Nuclei were stained with DAPI. Scale bar, 25 µm. (B) Immunofluorescence of FAM111B KO or WT RPE-1 cells mock-infected or infected with WT MCMV (MOI 1 TCID 50 /cell). Cells were fixed 40 hpi and stained with antibodies recognizing FAM111B (green) or MCMV E1 (red). Nuclei were stained with DAPI or Hoechst 33342. Arrow heads indicate FAM111B in vRCs. Scale bar, 25 µm.

Article Snippet: Polyclonal rabbit antibodies against FAM111B (HPA038637, Sigma-Aldrich), FAM111A (EPR14407, Abcam), Flag (F7425, Sigma-Aldrich), E2F3 (C-18, Santa Cruz) and E2F4 (C-20, Santa Cruz) were used.

Techniques: Immunofluorescence, Infection, Staining, Marker

Journal: bioRxiv

Article Title: Drosophila melanogaster Toll-9 elicits antiviral immunity against Drosophila C virus

doi: 10.1101/2024.06.19.599730

Figure Lengend Snippet: (A) In silico prediction of signal peptide in Toll-9 protein sequence. Red solid line indicates predicted n-terminal region, orange solid line indicates the predicted center hydrophobic region, and yellow solid line indicates predicted c-terminal region of signal peptide. Black dotted line indicates the cleavage site (CS) of the signal peptide. Sec/SPI: Sec translocon transported secretory signal peptide/Signal Peptidase I Tat/SPI: Tat translocon transported Tat signal peptides/Signal Peptidase I (B) Western blot analysis demonstrating the presence of Toll-9/V5 in endosomes. Endosomal fractions were identified using Rab5 as a microsomal marker, while Actin served as a cytosolic marker. Data are representative of three independent experiments. (C) Micrographs show colocalization of Rab5-early endosome marker (green) and Toll-9 (anti-V5 tag ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (D) Micrographs show colocalization of Rab7-Late endosome marker (green) and Toll-9 (anti-V5 tag ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (E) Micrographs show colocalization of Rab5-early endosome marker (green) and Poly(I:C) (J2 anti-dsRNA ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (F) Toll-9 (anti-V5 tag ab-Green) and Poly(I:C) (J2 anti-dsRNA ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (G) Western blot analysis using the indicated antibodies following immunoprecipitation of V5 tag (Toll-9) using J2 dsRNA antibody from the lysate of Poly (I:C) treated Toll-9 OE and S2 cells in presence and absence of CuSO 4 (500 µM). Data are representative from three independent experiments.

Article Snippet: The cells were blocked in phosphate-buffered saline (PBS) containing 10% FBS and incubated with antibodies against Rab5 (1:50; Abcam ab31261), Rab7(1:20; Developmental Studies Hybridoma Bank Rab7), DCV Capsid (1:200; Abcam ab92954), dsRNA J2 (1:200; Jena Biosciences RNT-SCI-10010200) and V5 tag (1:200; ThermoFisher Scientific R960-25) for overnight at 4°C.

Techniques: In Silico, Sequencing, Western Blot, Marker, Immunoprecipitation