Journal: Current biology : CB

Article Title: Modulation of Host Learning in Aedes aegypti Mosquitoes

doi: 10.1016/j.cub.2017.12.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Immunohistochemistry The polyclonal antiserum against tyrosine hydroxylase (ImmunoStar, Hudson, WI, USA - Cat. no. 22941) was used at a concentration of 1:50 and monoclonal antisera against synapsin I (Sigma-Aldrich, St. Louis, MO, USA - Cat. No. WH0006853M7) were used at a concentration of 1:100 for immunohistochemistry.

Techniques: Recombinant, Purification, CRISPR, Mutagenesis, Software

Journal: Cellular microbiology

Article Title: Enteropathogenic Escherichia coli effector EspF interacts with host protein Abcf2.

doi: 10.1111/j.1462-5822.2006.00820.x

Figure Lengend Snippet: Fig. 2. EspF interacts with Abcf2 in a yeast two-hybrid system. A. Plasmid pairs were co-transformed into yeast reporter strain AH109 and grown on minimal medium lacking leucine and tryptophan (SD-LT) to select for plasmid transformation. Transcriptional activation of reporter HIS3 was detected by plating onto minimal medium lacking leucine, tryptophan and histidine (SD-LTH), which specifically selects for protein interaction. B. The transcriptional activation of the non-selective reporter MEL1 was assessed in transformants by measuring the secreted a-galactosidase activity in SD-LT liquid medium. Error bars represent standard error from the mean of three experiments.

Article Snippet: Polyclonal antiserum against Abcf2 was produced by Proteintech (Chicago, IL).

Techniques: Plasmid Preparation, Transformation Assay, Activation Assay, Activity Assay

Journal: Cellular microbiology

Article Title: Enteropathogenic Escherichia coli effector EspF interacts with host protein Abcf2.

doi: 10.1111/j.1462-5822.2006.00820.x

Figure Lengend Snippet: Fig. 3. Abcf2 colocalizes partially with mitochondria in HeLa cells. Abcf2 was detected by indirect immunofluorescence with anti-Abcf2 affinity-purified antibodies (A) and mitochondria were labelled by prior transfection with a plasmid encoding DsRed2-MITO, a red fluorescent protein fused with a mitochondrial targeting signal (B). A single slice was acquired with a confocal microscope, the pinhole being set to achieve high Z-resolution (~0.5 mm). In the composite image (C), Abcf2 appears green, mitochondria appear red, coincident fluorescent sources appear orange-yellow. As a negative control, HeLa cells were co-transfected with a GFP vector (D) and DsRed2-MITO vector (E); GFP is excluded from mitochondria (composite image: panel F, compare red colour with orange seen in C). Scale bars correspond to 20 mm. (G) Cells were lysed and fractionated, Abcf2 and the mitochondrial marker cytochrome c (CYC) were detected by Western blotting. MH represents the ‘heavy mitochondrial’ fraction (3000 g pellet), ML represents the ‘light mitochondrial’ fraction (16 000 g pellet) and S represents the post-mitochondrial supernatant.

Article Snippet: Polyclonal antiserum against Abcf2 was produced by Proteintech (Chicago, IL).

Techniques: Transfection, Plasmid Preparation, Microscopy, Negative Control, Marker, Western Blot

Journal: Cellular microbiology

Article Title: Enteropathogenic Escherichia coli effector EspF interacts with host protein Abcf2.

doi: 10.1111/j.1462-5822.2006.00820.x

Figure Lengend Snippet: Fig. 4. EspF and Abcf2 can be co-immunoprecipitated from infected HeLa cells. HeLa cells were infected with espF mutant UMD874 or with UMD874 expressing EspF-FLAG from plasmid pJN61. The supernatants (S) and pellets (P) from immunoprecipitations carried out with antibodies to FLAG (first and second panels) or Abcf2 (third and fourth panels) and revealed with Abcf2 immunoblotting (first and fourth panels) or EspF immunoblotting (second and third panels) are shown. The positions of the relevant proteins are indicated. The exposure time for EspF was intentionally adjusted to reveal the faster migrating EspF band, which is a result of processing upon mitochondrial import (Nougayrède and Donnenberg, 2004).

Article Snippet: Polyclonal antiserum against Abcf2 was produced by Proteintech (Chicago, IL).

Techniques: Immunoprecipitation, Infection, Mutagenesis, Expressing, Plasmid Preparation, Western Blot

Journal: Cellular microbiology

Article Title: Enteropathogenic Escherichia coli effector EspF interacts with host protein Abcf2.

doi: 10.1111/j.1462-5822.2006.00820.x

Figure Lengend Snippet: Fig. 5. Silencing of Abcf2 by RNAi does not induce detectable cytotoxicity in HeLa cells. Cells were transfected twice over 48 h with Abcf2-specific siRNA or scrambled control siRNA, as indicated. Upper panels. F-actin was stained with rhodamine-phalloidin (red), DNA with DAPI (blue) and Abcf2 was revealed by immunofluorescence (green), using fixed imaging parameters for each sample. Bars correspond to 50 mm. Lower panels. Cells were analysed by flow cytometry, acquiring laser forward scatter (FSC channel) and indirect immunofluorescence for Abcf2 content (FL1 channel). Data from 10 000 cells are shown on bidimensional density plots. Laser side scatter (SSC channel) and cell cycle profiles according to DNA-content (PI staining and FL2 channel measurement) were also acquired but did not reveal differences between the three samples (not shown).

Article Snippet: Polyclonal antiserum against Abcf2 was produced by Proteintech (Chicago, IL).

Techniques: Transfection, Control, Staining, Imaging, Cytometry

Journal: Cellular microbiology

Article Title: Enteropathogenic Escherichia coli effector EspF interacts with host protein Abcf2.

doi: 10.1111/j.1462-5822.2006.00820.x

Figure Lengend Snippet: Fig. 6. Abcf2 depletion sensitizes HeLa cells to EspF and staurosporine-induced caspase cleavage. A. HeLa cells were transfected with Abcf2-specific siRNAs or scrambled control siRNA and then infected 3 h with a moi 100:1 of wild-type EPEC strain E2348/69, espF mutant strain UMD864 or UMD874 complemented with plasmid pJN61 that encodes FLAG-tagged EspF (espF + F) as shown. The cells were washed, lysed, proteins were separated by SDS-PAGE, blotted and probed with antibodies against Abcf2, cleaved caspase 9, cleaved caspase 3 (apoptosis markers), Hsc70 and actin (protein loading markers). B. Cells were transfected as in (A) and infected with wild-type EPEC. The mean relative content standard error from the mean, as measured by densitometry of Western blots (Abcf2 and cleaved caspase 9 relative to Hsc70 level) in four experiments is shown. C. Cells were transfected with scrambled or Abcf2-specific siRNAs. Then apoptosis was triggered by 4 h treatment with staurosporine (stauro), as shown. Cell lysates were probed as before for Abcf2, cleaved caspase 3 and actin. D. Cells were treated as in (C), and then the mean relative contents standard error from the mean of Abcf2 and cleaved caspase 3 as compared with actin in three experiments were measured by densitometry analysis of Western blots.

Article Snippet: Polyclonal antiserum against Abcf2 was produced by Proteintech (Chicago, IL).

Techniques: Transfection, Control, Infection, Mutagenesis, Plasmid Preparation, SDS Page, Western Blot

Journal: Cellular microbiology

Article Title: Enteropathogenic Escherichia coli effector EspF interacts with host protein Abcf2.

doi: 10.1111/j.1462-5822.2006.00820.x

Figure Lengend Snippet: Fig. 7. EPEC EspF decreases the amount of cellular Abcf2. HeLa cells were left uninfected or infected 3 h with various moi of (A) wild-type EPEC strain E2348/69, (B) espF mutant strain UMD874 or (C) UMD874 complemented with plasmid pJN61 encoding FLAG-tagged EspF (espF + F) as indicated, followed by Western blotting analyses for apoptosis marker cleaved caspase 9, Abcf2, EspF and protein loading control Hsc70. The upper panels represent the mean standard error from the mean densitometric measurements of Abcf2 and cleaved caspase 9 relative to Hsc70 signal, from three independent experiments. The lower panels show corresponding blots from one representative experiment.

Article Snippet: Polyclonal antiserum against Abcf2 was produced by Proteintech (Chicago, IL).

Techniques: Infection, Mutagenesis, Plasmid Preparation, Western Blot, Marker, Control

Journal: Cellular microbiology

Article Title: Enteropathogenic Escherichia coli effector EspF interacts with host protein Abcf2.

doi: 10.1111/j.1462-5822.2006.00820.x

Figure Lengend Snippet: Fig. 8. EPEC-induced caspase 9 cleavage and Abcf2 depletion require an intact T3SS. Confluent HeLa or Caco-2 cell monolayers were left uninfected, or infected 3 h with wild-type EPEC strain E2348/69, espF mutant strain UMD874, UMD874 complemented with plasmid pJN61 encoding FLAG-tagged EspF (espF + F), escN mutant strain CVD452, or CVD452 containing pJN61 (escN + F) at a moi of 200:1 followed by lysis and Western blotting of equal amounts of protein with anti-cleaved caspase 9, anti-Abcf2, anti-Hsc70 and anti-actin sera.

Article Snippet: Polyclonal antiserum against Abcf2 was produced by Proteintech (Chicago, IL).

Techniques: Infection, Mutagenesis, Plasmid Preparation, Lysis, Western Blot

Journal: The Journal of Cell Biology

Article Title: Identification of ERGIC-53 as an intracellular transport receptor of α 1 -antitrypsin

doi: 10.1083/jcb.200709100

Figure Lengend Snippet: Screening of the cDNA-YFP1 library for ERGIC-53 interaction partners. (A) COS-1 cells were transfected with the indicated bait and prey constructs and analyzed by FACS as described in Materials and methods. Coexpression of YFP2–ERGIC-53 and the cDNA-YFP1 library resulted in the specific detection of 1.63% YFP positive (YFP + ) cells. In a nonsaturating screen, ∼500 fluorescent cells were collected by FACS and total DNA was extracted and transformed into bacteria, which resulted in the recovery of several hundred prey clones. (B) 48 prey clones were randomly selected and plasmids were isolated and individually assayed by YFP PCA with YFP2–ERGIC-53 in COS-1 cells. Fluorometric analysis revealed that prey plasmids 17, 32, 33, and 44 (indicted by red boxes) were positive and reconstitute fluorescent YFP when expressed with YFP2–ERGIC-53. Bars represent fluorometric values of a single screening experiment. The threshold for a positive hit was set to 250 arbitrary fluorescence units, which corresponds to a ∼1.5-fold induction in YFP fluorescence in comparison to untransfected cells. (C) Sequence analyses identified α1-AT as a cDNA insert in all four positive prey plasmids.

Article Snippet: The following antibodies were used: mouse mAbs against human ERGIC-53 (G1/93; Qbiogene), human CLIMP-63 (G1/296; Qbiogene) and GFP (Roche), rabbit polyclonal antibodies (pAbs) against ERGIC-53 , human CLIMP-63, and horse fibronectin (provided by M. Chiquet, Friedrich Miescher Institute, Basel, Switzerland), goat pAb against human α1-AT (MP Biomedicals) and sheep pAb against human albumin (The Binding Site).

Techniques: Transfection, Construct, Transformation Assay, Clone Assay, Isolation, Fluorescence, Sequencing

Journal: The Journal of Cell Biology

Article Title: Identification of ERGIC-53 as an intracellular transport receptor of α 1 -antitrypsin

doi: 10.1083/jcb.200709100

Figure Lengend Snippet: ERGIC-53 captures α1-AT in a carbohydrate- and conformation-dependent manner. The indicated YFP PCA constructs were expressed in HeLa cells for 24 (A) and 48 h (B). 20 μM Lactacystin (lact) and 200 μM kifunensine (kif) were applied where indicated for 24 h. YFP complementation was analyzed by fluorometric analysis of cell suspensions in microtiter plates and expression levels of the different constructs were probed by Western blotting (WB) using polyclonal antibodies against ERGIC-53 and α1-AT. Endogenous ERGIC-53 functions as an input control. Bars represent mean ± SD ( n = 3).

Article Snippet: The following antibodies were used: mouse mAbs against human ERGIC-53 (G1/93; Qbiogene), human CLIMP-63 (G1/296; Qbiogene) and GFP (Roche), rabbit polyclonal antibodies (pAbs) against ERGIC-53 , human CLIMP-63, and horse fibronectin (provided by M. Chiquet, Friedrich Miescher Institute, Basel, Switzerland), goat pAb against human α1-AT (MP Biomedicals) and sheep pAb against human albumin (The Binding Site).

Techniques: Construct, Expressing, Western Blot

Journal: The Journal of Cell Biology

Article Title: Identification of ERGIC-53 as an intracellular transport receptor of α 1 -antitrypsin

doi: 10.1083/jcb.200709100

Figure Lengend Snippet: α1-AT secretion is impaired in ERGIC-53 knockdown cells. HepG2 cells were transiently transfected for 96 h with control and ERGIC-53–specific siRNA duplexes. (A) Western blotting using monoclonal anti–ERGIC-53 and anti–CLIMP-63 and polyclonal anti–α1-AT antibodies. ERGIC-53 is efficiently silenced, which results in intracellular accumulation of α1-AT. CLIMP-63, an ER-resident membrane protein, functions as an input control. (B) [ 35 S]methionine pulse chase of endogenous α1-AT and albumin. HepG2 cells were labeled for 15 min with [ 35 S]methionine and chased for the indicated times, and α1-AT and albumin were recovered by immunoprecipitation from cell lysates (intracellular) and conditioned medium (extracellular) using anti–α1-AT and anti-albumin antibodies, respectively. (C) The amount of intracellular and extracellular α1-AT and albumin was quantified by densitometric scanning of the band intensities and the secreted fraction of total protein [extracellular/(intracellular + extracellular)] was determined for each time point. Bars represent mean ± SD ( n = 3). Results analyzed by paired t test: NS, P > 0.05; **, P < 0.01.

Article Snippet: The following antibodies were used: mouse mAbs against human ERGIC-53 (G1/93; Qbiogene), human CLIMP-63 (G1/296; Qbiogene) and GFP (Roche), rabbit polyclonal antibodies (pAbs) against ERGIC-53 , human CLIMP-63, and horse fibronectin (provided by M. Chiquet, Friedrich Miescher Institute, Basel, Switzerland), goat pAb against human α1-AT (MP Biomedicals) and sheep pAb against human albumin (The Binding Site).

Techniques: Transfection, Western Blot, Pulse Chase, Labeling, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Identification of ERGIC-53 as an intracellular transport receptor of α 1 -antitrypsin

doi: 10.1083/jcb.200709100

Figure Lengend Snippet: ERGIC-53 is an intracellular transport receptor of α1-AT. α1-AT was transfected into wild-type (+/+) and ERGIC-53 knockout (−/−) MEFs and expressed for 24 h. (A) [ 35 S]methionine pulse chase of α1-AT and endogenous fibronectin. MEFs were labeled with [ 35 S]methionine for 15 min and chased for the indicated times, and α1-AT and fibronectin were recovered by immunoprecipitation from cell lysates (intracellular) and conditioned medium (extracellular) using anti–α1-AT and anti-fibronectin antibodies, respectively. (B) The secreted fraction of α1-AT and fibronectin was quantified as described in . (C) α1-AT was coexpressed with human ERGIC-53 in ERGIC-53 knockout MEFs (MEF −/− plus ERGIC-53). Western blotting was performed using polyclonal antibodies against ERGIC-53, α1-AT, and CLIMP-63. (D) [ 35 S]methionine pulse chase of α1-AT. (E) The secreted fraction of α1-AT after a 60-min chase was quantified by densitometric scanning and calculated as in [extracellular/(intracellular + extracellular)]. Bars represent mean ± SD ( n = 3). Results analyzed by paired t test: NS, P > 0.05; *, P < 0.05.

Article Snippet: The following antibodies were used: mouse mAbs against human ERGIC-53 (G1/93; Qbiogene), human CLIMP-63 (G1/296; Qbiogene) and GFP (Roche), rabbit polyclonal antibodies (pAbs) against ERGIC-53 , human CLIMP-63, and horse fibronectin (provided by M. Chiquet, Friedrich Miescher Institute, Basel, Switzerland), goat pAb against human α1-AT (MP Biomedicals) and sheep pAb against human albumin (The Binding Site).

Techniques: Transfection, Knock-Out, Pulse Chase, Labeling, Immunoprecipitation, Western Blot