Individual Sirna Duplexes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis"
Article Title: Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis
Journal: The Journal of Cell Biology
Figure Legend Snippet: The midbody positions the apical surface during cyst development. (A) Caco-2 transfected with control or Cdc42 siRNA was fixed and stained for DNA (blue), tubulin (green), and aPKC (red). (top) A control cyst at the two-cell stage (note that abscission appears to have occurred symmetrically). (middle) A larger control cyst, with the midbody in the center of the developing structure (apical region of dividing cell) reflecting asymmetric abscission. (bottom) Cdc42 siRNA structure with one midbody positioned normally at the center and another midbody (located in a different z section) abnormally positioned. (B) Quantitation of midbodies at the center of the cyst from three independent experiments. The total number of midbodies is indicated (N). A midbody is regarded as being in the center if it is located at a distance from the centroid that is less than one third the radius of the structure.
Techniques Used: Transfection, Staining, Quantitation Assay
Figure Legend Snippet: Cdc42 depletion disrupts mitotic spindle orientation. (A) Diagram depicting spindle angle measurement. The centroid of the cyst (dark blue circle) and the center of the spindle axis (pink circles) of a metaphase cell were drawn using ImageJ. The angle (red) between the spindle axis (black lines) and the line connecting the centroid of the cyst to the center of the spindle (dashed lines) was determined. To analyze spindle poles in different z sections, three z sections were taken so as to include both spindle poles and were merged as shown. Three schematic spindles are shown. The right and middle spindle examples represent correctly oriented spindles whose poles are positioned in one section (z2; middle spindle) or in different sections (z1 and z3; right spindle). The left spindle represents a misoriented spindle whose poles are in different z sections. Spindle microtubules, green; centrosomes, yellow; DNA, light blue. (B) Scatter diagram of metaphase spindle angles in cysts that were transfected with control or two Cdc42 siRNA duplexes from three independent experiments. Pink circles indicate mean values, green circles indicate individual data points, and error bars represent the SEM of the total number of spindles analyzed (N). (C) Caco-2 was transfected with control or Cdc42 siRNA and was fixed and stained for DNA (blue), tubulin (green), and filamentous actin (F-actin; red). Single confocal sections through the center of the cysts are shown. Three z sections are shown for the Cdc42 siRNA cyst to reveal both poles of the misoriented spindle. (D and E) Caco-2 transfected with control or Cdc42 siRNA was fixed and stained for DNA (blue) and aPKC (green). (D) Cdc42 siRNA structures contain cells in the middle, with apical domains present between inner and outer cells. (E) Cdc42 siRNA structures possess apical domains that are not in the center and single cells with more than one apical domain. Arrows indicate cells with multiple distinct apical patches on their surface. Bars, 10 μm.
Techniques Used: Transfection, Staining
Figure Legend Snippet: Cdc42 depletion induces multiple lumens. (A) Caco-2 was transfected with control or Cdc42 siRNA, plated in three dimensions, and treated with CTX at day 6 to induce luminal swelling. Phase images from cysts before (0 h) and after (12 h) treatment are shown. Note that about half of the Cdc42-depleted cysts lack a single central lumen. Bars, 50 μm. (B) Western blot of Caco-2 transfected with control siRNA, Cdc42 siRNA SMARTpool, or two different individual duplexes. Cdc42 levels are significantly reduced by 3 d and remain reduced for 7 d. (C) Caco-2 cultured as in A was fixed and stained with rhodamine phalloidin. Cysts were examined for single lumen (blue) or multiple lumens (red). The mean ± standard deviation for three independent experiments (at least 50 cysts each) is shown. (D and E) Caco-2 cultured as in A was fixed and stained for DNA (blue) and aPKC (green; D) or DNA (blue), E-cadherin (Ecad; green), and ZO-1 (red; E). Single confocal sections through the middle of the cysts are shown. DIC, differential interference contrast. Bars, 25 μm.
Techniques Used: Transfection, Western Blot, Cell Culture, Staining, Standard Deviation
2) Product Images from "Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions"
Article Title: Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions
Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology
Figure Legend Snippet: Downregulation of Aip1 expression increases paracellular permeability of colonic epithelial cell monolayers. SK-CO15 epithelial cells were transfected with different Aip1-specific siRNA duplexes and corresponding control siRNAs. On day 4 posttransfection, the efficiency of siRNA knockdown as well as the expression of Aip1-interacting cytoskeletal regulators and apoptosis markers was examined by electrophoresis and immunoblotting ( A ). ADF, actin-depolymerizing factor; PARP, poly(ADP-ribose) polymerase. Permeability of control and Aip1-depleted SK-CO15 cells was examined by measuring transepithelial electrical resistance (TEER) ( B ) and transepithelial flux of fluoresceinated dextrans ( C ). Data are presented as means ± SE ( n = 3); ** P
Techniques Used: Expressing, Permeability, Transfection, Electrophoresis
3) Product Images from "NOXA, a sensor of proteasome integrity, is degraded by 26S proteasomes by an ubiquitin-independent pathway that is blocked by MCL-1"
Article Title: NOXA, a sensor of proteasome integrity, is degraded by 26S proteasomes by an ubiquitin-independent pathway that is blocked by MCL-1
Journal: Cell Death and Differentiation
Figure Legend Snippet: 19S RP but not REG subunits are required for NOXA degradation. ( a ) Depletion of 19S RP subunits increases endogenous NOXA levels. HEK293T cells were transfected with siRNA pools (10 nM) against the indicated 19S RP or REG subunits for 48 h, and the indicated proteins were assessed by immunoblotting. REG β was undetectable in these cells, although REG β siRNA reduced REG α levels. ( b ) Depletion of 19S RP subunits inhibits 26S proteasome but increases 20S proteasome activity. HEK293T cells were transfected with the indicated siRNA pools (1 nM). Soluble extracts were subjected to 10–40% glycerol density gradient centrifugation and fractions assayed for Suc-LLVY-AMC hydrolyzing activity ± (0.02%). Fractions 1 and 22 correspond to the top (10%) and bottom (40%) of the gradient, respectively. Sedimentation positions of 20S and 26S proteasomes were assessed based upon co-fractionation with 20S CP subunits in the absence of 19S RP subunits or 20S CP subunits in the presence of 19S RP subunits, respectively. ( c ) MCL-1 is required to maintain endogenous NOXA levels, following depletion of the 19S RP subunit PSMC1. Cells were co-transfected with PSMC1 siRNA in the presence of a control or MCL-1 siRNA (10 nM). Indicated proteins were assessed by immunoblotting. ( d ) Proteasome inhibitor-induced accumulation of endogenous NOXA does not require MCL-1. Cells were transfected with either control or MCL-1 siRNA (1 nM) and immediately incubated with either DMSO, lactacystin (10 μ M) or epoxomicin (2.5 μ M) for 24 h. Indicated proteins were assessed by immunoblotting. ( e ) Degradation of NOXA-LL is blocked upon PSMC1 depletion. HEK293T cells were transfected for 24 h with control or PSMC1 siRNA (10 nM) and re-transfected for a further 24 h with empty vector, NOXA-WT or -LL. Degradation of NOXA was assessed by immunoblotting, following cycloheximide addition
Techniques Used: Transfection, Activity Assay, Gradient Centrifugation, Sedimentation, Fractionation, Incubation, Plasmid Preparation
Figure Legend Snippet: Inhibition of 26S proteasomes by depletion of the 19S RP subunit PSMD1 triggers NOXA-dependent apoptosis. ( a ) Apoptosis induced by depletion of different 19S RP subunits correlates with high endogenous NOXA levels. HeLa cells were transfected with siRNA pools of the indicated 19S RP subunits or a control siRNA (10 nM each) for 48 h and apoptosis quantified (top panel). Results are the means ±S.E.M. (n=3). Indicated endogenous proteins were assessed by immunoblotting (lower panel). ( b ) HeLa or ( c ) H1299 cells were transfected with a pool of PSMD1 siRNAs, and either a control or pool of NOXA siRNAs (10 nM) from Dharmacon (NOXA #1) or Ambion (NOXA #2). After 48 h, apoptosis was quantified (top panels). Results are the means ±S.E.M. (n=3). The levels of indicated proteins were assessed by immunoblotting (lower panels)
Techniques Used: Inhibition, Transfection
Article Title: Legionella pneumophila effector Lem4 is a membrane-associated protein tyrosine phosphatase
Article Snippet: .. The Orbitrap Elite raw files were searched against Homo sapiens proteins (taxonomy ID 9606; 20,983 Uniprot entries) and the Lem4-GFP sequence using the SequestHT algorithm in
Tandem Mass Spectroscopy:
Article Title: Proteomic analysis reveals different composition of extracellular vesicles released by two Trypanosoma cruzi strains associated with their distinct interaction with host cells
Article Snippet: .. Resulting MS/MS spectra from HR-MS/MS analysis of VF fractions of YuYu and Y strains were searched with Proteome Discoverer (PD)