Journal: PLoS ONE

Article Title: A no film slot blot for the detection of developing P . falciparum oocysts in mosquitoes

doi: 10.1371/journal.pone.0174229

Figure Lengend Snippet: Twofold serial dilutions of r Pf CSP protein were transferred to a nitrocellulose membrane using the slot blot apparatus and incubated with AP-conjugated C3103 mAb. Chemiluminescent signal from the membrane was captured using both autoradiography film and the C-Digit scanner. The resulting band intensities were fit to Michaelis-Menten regression curves to identify changes in the detection limit of the slot blot assay. Cutoff threshold intensities were determined from the mean intensity of negative control samples plus two standard deviations Film and the digital scan displayed similar sensitivities of approximately 0.01 pg of CSP. Intensities obtained on the C-digit scanner were generally bolder at lower intensities but declined more dramatically, with a steeper slope near the detection limit.

Article Snippet: We compared the sensitivity of the modified slot blot protocol using the C-Digit blot scanner to that of autoradiograph film in the detection of parasites from two independent populations of A . stephensi mosquitoes.

Techniques: Dot Blot, Incubation, Autoradiography, Slot Blot Assay, Negative Control

Journal: PLoS ONE

Article Title: A no film slot blot for the detection of developing P . falciparum oocysts in mosquitoes

doi: 10.1371/journal.pone.0174229

Figure Lengend Snippet: In three separate experiments performed on three different days, 0.25 oocyst was loaded into each slot blot well and data was captured with the C-digit blot scanner. Variability in band intensities from each day never exceeded 18% and overall inter-assay variability was estimated to be 9.85%.

Article Snippet: We compared the sensitivity of the modified slot blot protocol using the C-Digit blot scanner to that of autoradiograph film in the detection of parasites from two independent populations of A . stephensi mosquitoes.

Techniques: Dot Blot, Inter Assay

Journal: PLoS ONE

Article Title: A no film slot blot for the detection of developing P . falciparum oocysts in mosquitoes

doi: 10.1371/journal.pone.0174229

Figure Lengend Snippet: A batch of mosquitoes were fed an infectious blood meal with high percent Pf gametocytemia. On day 8 post feeding, a random sampling of 15 mosquitoes was dissected, stained, and analyzed using microscopy. Oocyst prevalence and intensity were estimated to be 86.7% and 20.5 oocysts per mosquito, respectively. This estimate was compared to the prevalence calculated from 20 different mosquitoes of the same batch that were homogenized and analyzed via slot blot data acquired using both film and the C-Digit blot scanner for data capture. Data obtained from both the film and blot scanner agreed very well, with 18/20 mosquitoes testing positive (90% prevalence) in both methods. The cutoff threshold for each analysis was determined by calculating the mean + 2SD of the band intensities of the negative or unfed mosquitoes.

Article Snippet: We compared the sensitivity of the modified slot blot protocol using the C-Digit blot scanner to that of autoradiograph film in the detection of parasites from two independent populations of A . stephensi mosquitoes.

Techniques: Sampling, Staining, Microscopy, Dot Blot

Journal: PLoS ONE

Article Title: A no film slot blot for the detection of developing P . falciparum oocysts in mosquitoes

doi: 10.1371/journal.pone.0174229

Figure Lengend Snippet: A batch of mosquitoes were fed an infectious blood meal with high percent Pf gametocytemia. On day 8 post feeding, a random sampling of 15 mosquitoes were dissected, stained, and analyzed using microscopy. Oocyst prevalence and intensity were estimated to be 20% and 1.4 oocyst per mosquito, respectively. This estimate was compared to the prevalence calculated from mosquitoes of the same batch that were homogenized and analyzed via slot blot data acquired using both film and the C-Digit blot scanner for data capture. Data obtained from both the film and blot scanner agreed very well, with 3/20 mosquitoes testing positive (15% prevalence) in both methods.

Article Snippet: We compared the sensitivity of the modified slot blot protocol using the C-Digit blot scanner to that of autoradiograph film in the detection of parasites from two independent populations of A . stephensi mosquitoes.

Techniques: Sampling, Staining, Microscopy, Dot Blot

Journal:

Article Title: Optimal Activation of an Endogenous Gene by HOX11 Requires the NH 2 -Terminal 50 Amino Acids

doi:

Figure Lengend Snippet: The Hep sequence can function as a sequence-specific activation domain. (A) The upper panel shows a yeast β-galactosidase slot blot assay of GAL4-DBD-Hep fusions. Fusions Y10 and Y12 to Y15 are represented schematically. The GAL4-DBD is fused to amino acids 1 to 50 of HOX11 (Y10), amino acids 16 to 30 of HOX11 (Y12), and amino acids 19 to 26 of HOX11 (Y13). Y14 and Y15 are based on Y12 but contain the amino acid substitutions shown. After incubation of the β-galactosidase assay mixture for 2 h, the ability of each fusion to activate the lacZ reporter gene was assessed by the blue color as indicated (when exposed for 12 h, Y14 and Y15 yielded blue coloration comparable to the 2-h exposure of Y12 and Y13). The lower panel shows Western blot detection of GAL4-DBD-HOX11 fusions in yeast extracts with a monoclonal GAL4-DBD antiserum. (B) The ability to activate in yeast is a conserved function of the Hep/octapeptide motif. The upper panel shows the results of a yeast β-galactosidase assay of GAL4-DBD-Hep/octapeptide fusions. The fusions Y12, Y16, and Y17 are represented schematically. GAL4-DBD is fused to a 15-amino-acid peptide encompassing the HOX11 Hep motif (Y12), the Hlx Hep motif (Y16), or the octapeptide sequence from Pax-2 (Y17). The lower panel shows Western blot detection of GAL4-DBD-Hep/octapeptide fusions in yeast extracts with a monoclonal GAL4-DBD antiserum.

Article Snippet: The membranes were blocked for 1 h in 5% Marvel (Premier Beverages) solution and incubated with either a monoclonal GAL4-DBD antiserum (Santa Cruz Biotechnology) or a polyclonal HOX11 antiserum (see below).

Techniques: Sequencing, Activation Assay, Slot Blot Assay, Incubation, Western Blot

Journal:

Article Title: Optimal Activation of an Endogenous Gene by HOX11 Requires the NH 2 -Terminal 50 Amino Acids

doi:

Figure Lengend Snippet: The NH2-terminal region of HOX11 can activate transcription in mammalian cells. (A) CAT assay of Cos-7 cells after transfection of GAL4-DBD-HOX11 fusion plasmids. Cos-7 cells were transfected with the pG5EC reporter construct alone or in combination with various GAL4-DBD expression vectors: pM1 (expressing the GAL4-DBD alone), pM1-N98 (expressing amino acids 1 to 98 of HOX11 fused to the GAL4-DBD), pM1-N50 (expressing amino acids 1 to 50 of HOX11 fused to the GAL4-DBD), pM1-N50ΔHEP (based on pM1-N50 but containing an internal deletion of the Hep motif at amino acids 19 to 26), or pVP65 (expressing the VP65 activation domain fused to the GAL4-DBD). (B) Western blot detection of GAL4-DBD-HOX11 fusions in Cos-7 cell extracts. Extracts were prepared 36 h after transfection and analyzed with a monoclonal GAL4-DBD antiserum. GAL4-DBD fusion proteins were detected more readily than was GAL4-DBD, for unknown reasons. (C) Chart showing the relative CAT activities of pM1-N98, pM1-N50, and pM1-N50ΔHEP after normalization for protein expression levels. CAT activity and protein levels were quantitated by densitometry. The values are expressed as percentages, with pM1-N50 activity assigned the value of 100%.

Article Snippet: The membranes were blocked for 1 h in 5% Marvel (Premier Beverages) solution and incubated with either a monoclonal GAL4-DBD antiserum (Santa Cruz Biotechnology) or a polyclonal HOX11 antiserum (see below).

Techniques: Transfection, Construct, Expressing, Activation Assay, Western Blot, Activity Assay

Journal:

Article Title: Optimal Activation of an Endogenous Gene by HOX11 Requires the NH 2 -Terminal 50 Amino Acids

doi:

Figure Lengend Snippet: Yeast one-hybrid assay of HOX11 transcriptional activation domains. GAL4-DBD-HOX11 fusions are represented schematically on the left. The position of the homeodomain is indicated by the dashed vertical lines, and mutants lacking the third helix of the homeodomain (amino acids 242 to 251) are marked by the symbol ΔH3. The ability of each fusion to activate the lacZ reporter gene, as assessed by the presence of a blue color in the slot blot β-galactosidase assay, is indicated on the right. Apparent differences in the intensity of blue coloration are ignored because although expression of the GAL4-DBD alone and fusions Y1 to Y11 was confirmed by Western blot analysis with a monoclonal GAL4-DBD antiserum, the levels of protein expression varied (data not shown).

Article Snippet: The membranes were blocked for 1 h in 5% Marvel (Premier Beverages) solution and incubated with either a monoclonal GAL4-DBD antiserum (Santa Cruz Biotechnology) or a polyclonal HOX11 antiserum (see below).

Techniques: Y1H Assay, Activation Assay, Dot Blot, Expressing, Western Blot

Journal:

Article Title: Compensatory Point Mutations in the Human Immunodeficiency Virus Type 1 Gag Region That Are Distal from Deletion Mutations in the Dimerization Initiation Site Can Restore Viral Replication

doi:

Figure Lengend Snippet: Viral RNA packaging in wild-type and mutated viruses. (A) Analysis of viral RNA packaging by slot blotting. Viral particles were harvested from culture fluids of transfected COS-7 cells and centrifuged through a 20% sucrose cushion. Viral RNA was extracted and dissolved in double-distilled water to a concentration equivalent to 8 ng of p24 (CA) antigen per μl. After treatment with RNase-free DNase I, RNA samples were subjected to slot blotting. As a control, samples were digested with RNase A to rule out possible DNA contamination. Relative RNA content was quantified by molecular image analysis. Levels of RNA in wild-type HIV-1 were arbitrarily set at 1.0. (B) Analysis of viral RNA packaging by RT-PCR. Reverse transcription was performed by using AMV RT to extend viral RNA that had been annealed to DNA primer pST. Reverse transcription products were amplified in a 15-cycle PCR by using primer pair GAG1/pST to generate a 119-bp DNA fragment. Relative amounts of DNA products were quantified by molecular imaging, with wild-type levels arbitrarily set at 1.0. Reactions run without RT served as a negative control to exclude any potential DNA contamination. As a positive control, the 15-cycle PCR was also performed with 5 ng of proviral BH10 DNA.

Article Snippet: For slot blot assays, 10 μl of viral RNA samples was treated with 10 U of DNase I (RNase-free; GIBCO BRL) at 37°C for 10 min and then heated to 95°C for 10 min to inactivate this enzyme.

Techniques: Transfection, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Imaging, Negative Control, Positive Control

Journal: Cell reports

Article Title: The FANCI/FANCD2 complex links DNA damage response to R-loop regulation through SRSF1-mediated mRNA export

doi: 10.1016/j.celrep.2023.113610

Figure Lengend Snippet: (A) Genomic DNA (0.6 mg) from FA-D2, FA-D2+FANCD2, and FA-D2+K561R cells was extracted, left untreated or treated with RNase H at 37°C for 1 h, and analyzed with slot blot assay using the S9.6 antibody. Methylene blue staining was used as a loading control (left). Densitometry of the slot blot shows the S9.6 intensity normalized to FA-D2+FANCD2 (right). (B) HeLa cells transfected with siCTRL, siSRSF1, siFANCD2, or siSRSF1+siFANCD2 double knockdown were subjected to immunofluorescence to visualize RNA:DNA hybrids using the S9.6 antibody. RNAseH1 treatment was used as a control for S9.6 antibody specificity. Samples were co-stained with nucleolin to subtract the nucleolar S9.6 signal (top). The scatterplot shows the quantification of S9.6 intensity per nucleus after subtraction of the nucleolar signal. At least 65 cells per slide were analyzed (bottom). (C) Genomic DNA from samples in (A) (0.3 or 0.6 μg) was left untreated or treated with RNAseH1 at 37°C for 1 h and analyzed with S9.6 slot blot. Methylene blue staining was used as a loading control (left). Densitometry of the slot blot shows the S9.6 intensity normalized to siCTRL (right). (D) Schematic of the DART system (left). Immunostaining of SRSF1 was done in four U2OS TRE cell lines expressing different fusion effector proteins in a DART assay (center). Quantification of the average SRSF1 foci intensity of at least 50 cells per condition was performed (right). (E) TA-KR-transfected U2OS TRE cells showing the colocalization of FANCD2 and SRSF1. (F) siRNA depletion of FANCD2, SRSF1, or AQR increased S9.6 focus intensity in a DART assay (left). Shown is quantification of the average S9.6 focus intensity of at least 50 cells per condition (right). Data represent the mean and SEM of three independent experiments. Statistics were performed using the unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.00, ****p < 0.0001. See also .

Article Snippet: ShortCut RNase III , NEB , Cat# M0245S.

Techniques: Slot Blot Assay, Staining, Control, Dot Blot, Transfection, Knockdown, Immunofluorescence, Immunostaining, Expressing

Journal: Cell reports

Article Title: The FANCI/FANCD2 complex links DNA damage response to R-loop regulation through SRSF1-mediated mRNA export

doi: 10.1016/j.celrep.2023.113610

Figure Lengend Snippet: (A) Immunoblot of HeLa cells co-transfected with siCTRL or siSRSF1 as well as RNase H1-GFP or GFP-N1 empty vector and untreated or treated with 1 μM MMC for 24 h. Ku86 was used as a loading control (left). Densitometry of the immunoblot shows ub-FANCD2/FANCD2. The data represent the mean and SEM of three independent experiments. Data were analyzed using the unpaired t test (right). (B) Same cells from (A) were subjected to immunofluorescence using FANCD2 antibody. The graph shows the quantification of FANCD2 foci. Data (mean and SEM) are representative of three independent analyses of at least 50 cells per slide. Data were analyzed using the unpaired t test. (C) HeLa cells were treated with the indicated concentrations of MMC, and cell survival was analyzed by crystal violet staining after incubation at 37°C for 4 days. The percentages of surviving cells were normalized to the untreated control and are shown as the mean and SEM of three independent experiments. Data were analyzed using two-way ANOVA. *, compared with siCTRL; #, compared with siSRSF1+RNAseH1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also .

Article Snippet: ShortCut RNase III , NEB , Cat# M0245S.

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Immunofluorescence, Staining, Incubation

Journal: Cell reports

Article Title: The FANCI/FANCD2 complex links DNA damage response to R-loop regulation through SRSF1-mediated mRNA export

doi: 10.1016/j.celrep.2023.113610

Figure Lengend Snippet: (A) HeLa cells were transfected with mock or FLAG-SRSF1 and treated or not with RNAse A. Whole-cell extracts were immunoprecipitated with FLAG-M2 agarose, and immunoprecipitation products were analyzed for SRSF1 interaction with FANCD2 and NXF1 antibodies. (B) Whole-cell extracts from FA-D2 mutant cells, FA-D2+FANCD2, and FA-D2+K561R cells were prepared and immunoprecipitated with IgG or SRSF1 antibody. The immunoprecipitation products were analyzed for SRSF1 interaction with NXF1 antibody. (C) Whole-cell extracts from FA-D2+FANCD2 cells were treated or not with increasing concentrations of MMC for 24 h and immunoprecipitated with IgG or SRSF1 antibody. The immunoprecipitation products were analyzed for SRSF1 interaction with NXF1 antibody. (D) Schematic of the approach followed to detect binding of FANCD2, NXF1, and SRSF1 to mature RNA by appending MS2 trap (6× MS2 stem loops) at the end of the 3′ UTR of targets, creating a downstream chimeric RNA luciferase (left). Immunoblotting was used to detect RNA binding proteins (RBPs) by FLAG-MS2-BP immunoprecipitation. β-Actin was used as a control for loading and to assess specificity of the immunoprecipitation (IP). (E) Immunoblot showing FLAG-MS2-BP immunoprecipitation in FA-D2 mutant, FA-D2+FANCD2, and FA-D2+K561R cells. β-Actin was used as control for loading and to assess the specificity of the IP. (F) FA-D2 mutant, FA-D2+FANCD2, and FA-D2+K561R cells showing the translocation of poly(A) RNAs detected by RNA FISH (oligo-dT probe, red). Using image analyses, the poly(A) signal in the nucleus and cytoplasm was quantified to calculate the nuclear/cytoplasmic (N/C) ratio. Each circle in the graph represents the mean of the poly(A) N/C ratio of at least 15 cells, and the mean ( ± SEM) is also indicated. The data correspond to three independent experiments and were analyzed using the unpaired t test (right). (G) Graph showing the N/C ratio of the mRNA targets identified by RNA-seq in FA-D2, FA-D2+FANCD2, and FA-D2+K561R cells. Each circle in the graph represents the N/C value of a specific mRNA target. Data were analyzed using unpaired t test compared with FA-D2+FANCD2 wild-type cells. *p < 0.05, **p < 0.01, ***p < 0.00, ****p < 0.0001. See also .

Article Snippet: ShortCut RNase III , NEB , Cat# M0245S.

Techniques: Transfection, Immunoprecipitation, Mutagenesis, Binding Assay, Luciferase, Western Blot, RNA Binding Assay, Control, Translocation Assay, RNA Sequencing Assay

Journal: Cell reports

Article Title: The FANCI/FANCD2 complex links DNA damage response to R-loop regulation through SRSF1-mediated mRNA export

doi: 10.1016/j.celrep.2023.113610

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ShortCut RNase III , NEB , Cat# M0245S.

Techniques: Purification, Virus, Recombinant, Agarose Gel Electrophoresis, Staining, Mutagenesis, Control, In Vitro, Binding Assay, Software