Journal: PLoS ONE

Article Title: Studying the Salt Dependence of the Binding of ?70 and ?32 to Core RNA Polymerase Using Luminescence Resonance Energy Transfer

doi: 10.1371/journal.pone.0006490

Figure Lengend Snippet: The effect of common buffer components on the LRET assay. Common buffer components were tested for their ability to alter the interaction 10 nM Tb-core with 20 nM F-σ70 after a 1-hour incubation at 22°C. The average value of A 520 /A 490 for three replicates was determined and normalized to a no-treatment control with a buffer containing 50 mM Tris-HCl pH 7.9, 5% glycerol, and 100 mM NaCl. The error bars represent the normalized standard deviation for a sample size of 4. A) Common solvents (DMSO, ethanol, and methanol) were tested in a range of 0–50% (v/v). B) The non-ionic detergents TritonX-100 and Tween-20 were tested from 0–5% (v/v). C) Glycerol was tested from 2.5–50% (v/v). The minimum glycerol level (2.5%) was due to the glycerol in the buffer of the proteins. D) Bovine serum albumin (BSA) and MgCl 2 were tested from 0–1000 µg/mL and 0–500 mM, respectively.

Article Snippet: Non-ionic detergents such as TritonX-100 and Tween-20 had no effect up to 2.5% ( ).

Techniques: Incubation, Standard Deviation

Journal: PLoS ONE

Article Title: Construction of DNA Hemicatenanes from Two Small Circular DNA Molecules

doi: 10.1371/journal.pone.0119368

Figure Lengend Snippet: Effect of Triton X-100 on the migration of single stranded DNA in polyacrylamide gels. The gel illustrates the smearing of single stranded DNA when loaded on polyacrylamide gels in the presence of Triton X-100. The DNA sample was the same in all lanes and contained a labeled single stranded circle of 235 nucleotides. Two minor bands that show no smearing most probably correspond to contaminating double stranded DNA, as smearing was always observed with many different single strands. Three different brands of Triton-X100 denoted T1–T3 were tested at a final concentration of 0.05%: T1 and T2 were ordinary grade, T3 was highly purified, oxidant-free Triton-X100, sold in glass ampules sealed under nitrogen (Pierce). Lane 1: control, no Triton-X100 added; lane 2: deionized T1; lane 3: T1, untreated; lane 4: T1, heat-treated; lane 5: T1, SDS added at a concentration of 0.005%; lane 6: T3, untreated; lane 7: T1, washed with a 5M NaCl solution; lane 8: T2, untreated. Note that addition of SDS to the samples before loading the gel was a very efficient way to suppress smearing (lane 5).

Article Snippet: Deionization of Triton-X100 with mixed-bed ion exchange resin, or use of highly-purified Triton-X100 sold in glass ampules sealed under nitrogen (Pierce), did not significantly change the result ( ).

Techniques: Migration, Labeling, Concentration Assay, Purification

Journal: Molecular Cancer

Article Title: TGF?1 activates c-Jun and Erk1 via ?V?6 integrin

doi: 10.1186/1476-4598-2-33

Figure Lengend Snippet: Erk1 is associated with focal adhesions. BxPC-3 cells were stimulated with 10 nM of mature TGFβ 1 for ten minutes followed by preparation of the Triton-X100 nonsoluble fraction and precipitation with α V β 6 integrin antibodies. The precipitate was then re-precipitated with anti-FAK antibodies (sc-1688) and analyzed with an Erk1 antibody (sc-93). In part, the cells were preincubated with a TGFβ 1 antibody [ 10 ].

Article Snippet: After preparation of the cytoskeletal fraction by Triton-X100 extraction [ , , ], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for Actin, Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αV β6 integrin, and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 labeling [ ].

Techniques:

Journal: Molecular Cancer

Article Title: TGF?1 activates c-Jun and Erk1 via ?V?6 integrin

doi: 10.1186/1476-4598-2-33

Figure Lengend Snippet: Paxillin and c-Jun are associated with focal adhesions. BxPC-3 cells were stimulated with 10 nM of mature TGFβ 1 for ten minutes followed by preparation of the Triton-X100 nonsoluble fraction and precipitation with α V β 6 integrin antibodies. The precipitate was then re-precipitated with anti-FAK antibodies (sc-1688) and analyzed with antibodies against p-Paxillin Y118 (2541, Cell Signaling) and p-c-Jun S63 (9621, Cell Signaling). In part, the cells were preincubated with a TGFβ-RII antibody (Santa Cruz), BAPTA-AM and Cytochalasin D, respectively [ 10 ].

Article Snippet: After preparation of the cytoskeletal fraction by Triton-X100 extraction [ , , ], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for Actin, Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αV β6 integrin, and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 labeling [ ].

Techniques:

Journal: Molecular Cancer

Article Title: TGF?1 activates c-Jun and Erk1 via ?V?6 integrin

doi: 10.1186/1476-4598-2-33

Figure Lengend Snippet: MEKK1 is associated with focal adhesions. BxPC-3 cells were stimulated with 10 nM of mature TGFβ 1 for ten minutes followed by preparation of the Triton-X100 nonsoluble fraction and precipitation with α V β 6 integrin antibodies. The precipitate was then re-precipitated with anti-FAK antibodies (sc-1688) and analyzed with a MEKK1 antibody (sc-448). In part, the cells were preincubated with a TGFβ 1 antibody [ 10 ].

Article Snippet: After preparation of the cytoskeletal fraction by Triton-X100 extraction [ , , ], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for Actin, Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αV β6 integrin, and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 labeling [ ].

Techniques:

Journal: Molecular Cancer

Article Title: TGF?1 activates c-Jun and Erk1 via ?V?6 integrin

doi: 10.1186/1476-4598-2-33

Figure Lengend Snippet: Pak1 is associated with focal adhesions. BxPC-3 cells were stimulated with 10 nM of mature TGFβ 1 for ten minutes followed by preparation of the Triton-X100 nonsoluble fraction and precipitation with α V β 6 integrin antibodies. The precipitate was then reprecipitated with anti-FAK antibodies (sc-1688), followed by re-precipitation with anti-phospho S (ab9334, Abcam), anti-phospho Thr (ab2286), and analyzed with a Pak1 antibody (71-9300, Zymed). In part, the cells were preincubated with 50 μM PP2 (Calbiochem), and 50 μM PD98059 (Calbiochem), respectively [ 10 ].

Article Snippet: After preparation of the cytoskeletal fraction by Triton-X100 extraction [ , , ], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for Actin, Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αV β6 integrin, and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 labeling [ ].

Techniques:

Journal: Molecular Cancer

Article Title: TGF?1 activates c-Jun and Erk1 via ?V?6 integrin

doi: 10.1186/1476-4598-2-33

Figure Lengend Snippet: Colocalization of TGFβ 1 , α V β 6 integrin and the cytoskeleton. 10,000 cells(SW48, DLD1, HeLa, keratinocytes) were cultured on glass coverslips in DMEM supplemented with 17% of heat inactivated fetal bovine serum and stimulated with 10 nM of mature TGFβ 1 (from R D Systems) for ten minutes. After preparation of the cytoskeletal fraction by Triton-X100 extraction [ 10 ], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for actin (sc-8432, Santa Cruz), Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for α V β 6 integrin (sc-6617 and sc-6632), and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ 1 (sc-146) labeling and viewed using a Zeiss LSM-510 confocal microscope [ 10 ]. Magnification 1000 ×.

Article Snippet: After preparation of the cytoskeletal fraction by Triton-X100 extraction [ , , ], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for Actin, Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αV β6 integrin, and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 labeling [ ].

Techniques: Cell Culture, Staining, Labeling, Microscopy

Journal: PLoS ONE

Article Title: An Essential Role of the Universal Polarity Protein, aPKC?, on the Maintenance of Podocyte Slit Diaphragms

doi: 10.1371/journal.pone.0004194

Figure Lengend Snippet: PAR3 forms a complex with aPKC, nephrin, and podocin in vivo . (A) The distribution of PAR3, aPKCλ, nephrin, and podocin was analyzed in differentially extracted fractions of rat renal tubules or glomeruli (Tub, Gl). Tub-S1, Gl-S1, highly soluble fractions extracted with 0.1% TritonX-100; Tub-S2, Gl-S2, relatively soluble fractions extracted with 1.0% TritonX-100 and 20 mM CHAPS; Tub-P2, Gl-P2, insoluble fractions. The blotted filter with anti-nephrin antibody was stripped and reprobed with anti-PAR3 antibody. (B) Immunoprecipitation for PAR3 was carried out on both glomerular fractions (Gl-S1, Gl-S2) using two independent anti-PAR3 antibodies (UBI, C2-2AP) and the immunoprecipitates were analyzed by immunoblotting. The blotted filter with anti-nephrin antibody was stripped and reprobed with anti-PAR3 antibody.

Article Snippet: GST pull-down assay Purified GST fusion proteins were separately incubated for 2 h at 4°C with 293T cells expressing the PAR3 proteins extracted with 25 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM EDTA, 1% TritonX-100, and protease inhibitor cocktail (Sigma), or with purified MBP-fusion proteins in 20 mM HEPES–NaOH (pH 7.5), 100 mM NaCl, 5 mM EDTA, 1% TritonX-100, 0.5 mg/ml bovine serum albumin, and 10% glycerol.

Techniques: In Vivo, Immunoprecipitation

Journal: Traffic (Copenhagen, Denmark)

Article Title: Alphavirus Restriction by IFITM Proteins

doi: 10.1111/tra.12416

Figure Lengend Snippet: IFITM3 expression inhibits SFV capsid release . A) SFV (200 pfu/cell) was bound to cells for 1 h at 4°C prior to incubation at 37°C for the indicated times. At each time point the cells were fixed and permeabilized with 0.1% Triton‐X100 and labeled with serum against the SFV capsid, which was detected with AF594. Confocal sections are displayed. Following internalization, virus particles were detected in cells, indicated by the typical punctate association of virus with endosomes. By 40 min after warm‐up diffuse cytosolic fluorescence was seen in A549 cells, indicating release of the viral capsid protein to the cytosol. In A549 cells treated with 10 µ m monensin, and in OS‐IFITM3‐HA expressing cells, the staining remains associated with puncta and the cytosolic staining was not observed even at 60 min after warm‐up. Nuclei were detected with Hoechst staining. Scale bar represents 15 µm. B) Quantification of cytosolic fluorescence and significance testing is described in Materials and Methods . The data are from three independent infections with 3–7 images taken at each condition, with a total of at least 60 cells analyzed per cell line, per condition. Data presented is the average fluorescence intensity, normalized to the background intensity at 0 min of warming. *p

Article Snippet: Immunofluorescence staining and microscopy and flow cytometry Immunofluorescence staining and microscopy was performed as previously described with the exception that 0.1% Triton‐X100 (Tx100, Sigma) was used for permeabilization in experiments to detect the SFV capsid protein.

Techniques: Expressing, Incubation, Labeling, Fluorescence, Staining

Journal: PLoS ONE

Article Title: Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

doi: 10.1371/journal.pone.0191052

Figure Lengend Snippet: Membrane expression and functionality of hLDLR expressed by the CHO-hLDLR-EGFP cell line. (A) Representative confocal photomicrographs of immunostained CHO-hTfR-EGFP (negative control) and CHO-hLDLR-EGFP cells (green) with an anti-LDLR antibody diluted at 1/100 (red) following triton X100 permeabilization of the cell membrane. Cell nuclei are labeled with Hoechst#33258 at 0.5 μg/mL (blue). Co-labeling appears in yellow in the merged pictures. Only the cells with stable expression of the hLDLR-EGFP construct express the membrane receptor. (B) Representative confocal photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) after a 10 min incubation period with DiI-LDL 15 μg/mL (red). Cell nuclei are labeled with Hoechst#33258 (blue). Co-labeling appears in yellow in the merged pictures. DiI-LDL is essentially bound to the CHO-hLDLR-EGFP cells indicating that the hLDLR-EGFP chimera receptor binds its ligand. (C) Western blots performed on cell membrane preparations of CHO cells expressing hLDLR-EGFP, rLDLR-EGFP and mLDLR-EGFP compared to CHO WT, using anti-hLDLR (1/800) and anti-rat and mouse LDLR antibodies (1/1000). These bands are also labeled with an anti-EGFP antibody. Bands of 140 kDa and 190 kDa correspond respectively to the immature and mature LDLR-EGFP fusion proteins (arrows). Actin was used to check loading of equal amounts of protein.

Article Snippet: In order to detect the Fc conjugates in tissues of injected mice, organs (liver and adrenals), were mechanically dissociated in PBS 0.1% Triton X100, snap frozen, thawed and sonicated 10 x 10 s. Protein content in membrane and tissue extracts were quantified using the BioRad DC Protein Assay (Bio-Rad, Hercules, CA, USA) following manufacturer’s instructions.

Techniques: Expressing, Negative Control, Labeling, Construct, Incubation, Western Blot