r0189  (New England Biolabs)

Journal: Autophagy

Article Title: Features of autophagic cell death in Plasmodium liver-stage parasites

doi: 10.4161/auto.23689

Figure Lengend Snippet: Figure 7. PbAtg8 does not localize to autophagosomes in dying parasites ( A ) and cannot complement yeast Atg8 ( B ). ( A ) HepG2 cells were infected with P. berghei parasites constitutively expressing mCherry (mCherry). 60 hpi the cells were fixed and stained with an anti-PbACP antiserum (ACP) to label the apicoplast and an anti-PbAtg8 antiserum (PbAtg8) to monitor the localization of this protein during parasite cell death. DNA was labeled with DAPI. A representative parasite showing strong vacuolization is depicted. A higher magnification of some important details is presented in the merged image. Scale bar: 10 µm: CPS. ( B ) Scatg8Δ and wt strains were transformed with empty pFL61 plasmid or the same plasmid containing PbAtg8 or ScAtg8 as a positive control. Western blot analysis was performed with the transformed strains using anti-aminopeptidase I antibodies. Transport of prApe1 to the vacuole where it matures (Ape1) only takes place in the presence of a functional autophagy pathway. The prApe1 and the Ape1 bands are marked with arrows.

Article Snippet: The fragments were cloned into the yeast expression vector pFL61 via NotI (New England Biolabs, R0189) and the resulting plasmids used to transform Scatg8Δ WCG strains of S. cerevisiae by the acetate method.

Techniques: Infection, Expressing, Staining, Labeling, Transformation Assay, Plasmid Preparation, Positive Control, Western Blot, Functional Assay

Journal: bioRxiv

Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

doi: 10.1101/2020.07.02.184408

Figure Lengend Snippet: Generation of synthetic α 21-I TetO and α 21-II LacO/Gal4 arrays. (A) Scheme of the pBAC11.32TW12.32GLII containing BAC and YAC cassettes, G418 resistance cassette and synthetic DNA: α 21-I TetO formed by high ordered repeats (HOR) monomers (green arrows) containing CENP-B boxes (blue) alternate with monomers containing TetO (yellow); α 21-II LacO/Gal4 formed by high ordered repeats (HOR) monomers (yellow arrows) containing Gal4 binding sequence (green) alternating with LacO (red). (B) Schematic of the assembly of the α 21-I TetO and α 21-II LacO/Gal4 arrays. (C, D) PFGE analysis of the nascent α 21-I TetO and α 21-II LacO/Gal4 arrays, cut with BamHI/NotI after each cycles of tandem ligation array amplification as described in Figure S2A (C) and Figure S2B (D). Expected sizes: α 21-I TetO 11-mer 1 copy (1.9 kb), 8 copies (15.2 kb), 32 copies (60.8 kb); α 21-II LacO/Gal4 12-mer 1 copy (2 kb), 8 copies (16 kb), 32 copies (64 kb). Plasmid vector is 2.9 kb, BAC vector is 7.1 kb. The asterisk (*) indicates the fragments that have been cloned into BAC vector (8 copies, 16 kb); red arrow in D indicates the size of the final pBAC11.32TW12.32GLII (∼120 kb) (m and M, markers).

Article Snippet: After each cloning step, the forming arrays were digested with BamHI and NotI restriction enzymes (NEB) and analysed on 1% agarose gel electrophoresis using 100 bp DNA Ladder, Quick-Load 1 kb Extend DNA ladder or Low Range PFG Marker (NEB).

Techniques: BAC Assay, Binding Assay, Sequencing, Ligation, Amplification, Plasmid Preparation, Clone Assay

Journal: bioRxiv

Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

doi: 10.1101/2020.07.02.184408

Figure Lengend Snippet: Screening of HT1080 colonies after transfection with pBAC11.32TW12.32GLII. (A) Scheme showing the possible fates of the pBAC11.32TW12.32GLII HAC seeding DNA after transfection in HT1080: in yellow and green (as integration or HAC) is represented the synthetic DNA. Timeline of the experiments performed from transfection into HT1080 cells. (B) BAC copy number (y axis) analyzed by qPCR in each HT1080 clone (x axis): only HT1080 clones containing > 20 BAC copies are represented in the graph. HT1080 clones are represented in green (HAC), red (integration) or mixture (both) according to the results of the FISH screening, as shown in C. Black arrows indicate the clones shown in C and analyzed further. (C) Representative pictures of oligo-FISH staining of HT1080 clones: slides have been hybridized with DNA probes (TetO-dig/rhodamine -dig antibody, Gal4-biotin and LacO-biotin/Fitc-streptavidin). DAPI stains DNA. Scalebar = 10µm. (D) Southern blot of selected HT1080 clonal DNA (as labelled on top of the panel) digested with BamHI and separated by CHEF; the transferred membrane was hybridized with radioactively labelled TetO (left) or LacO (right) specific probes. Red arrows indicate the expected size of the band without rearrangements. Clones labelled in red have been screened further (M and m, markers). (E) Cartoon of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI.

Article Snippet: After each cloning step, the forming arrays were digested with BamHI and NotI restriction enzymes (NEB) and analysed on 1% agarose gel electrophoresis using 100 bp DNA Ladder, Quick-Load 1 kb Extend DNA ladder or Low Range PFG Marker (NEB).

Techniques: Transfection, HAC Assay, BAC Assay, Real-time Polymerase Chain Reaction, Clone Assay, Fluorescence In Situ Hybridization, Staining, Southern Blot

Journal: bioRxiv

Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

doi: 10.1101/2020.07.02.184408

Figure Lengend Snippet: Formation of input pBAC11.32TW12.32GLII DNA. (A) CHEF analysis of 16 bacterial DNA after transformation with pBAC11.32TW12.32GLII and NotI and BamHI digestion: red arrows indicate the size of the final vector (∼120 kb); colonies labelled in red contain the insert of the desired length. DNA used for transfection as a control (in duplicate) (M marker). (B) Scheme of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI used to release the synthetic DNA. (C) PFGE analysis of selected bacterial colonies (in red) digested with EcoRI: each fragment originates from a different array (label on the left). DNA used for transfection as a control (in duplicate); original DNA as uncut sample (M marker). (D) α 21-I TetO and α 21-II LacO/Gal4 DNA ratio calculated with ImageJ on the intensity of the bands shown in C for each bacterial colony. Control and original DNA as in C. (E) CHEF analysis of bacterial colony #1 DNA (in duplicate) digested with NotI and BamHI to release the synthetic DNA (m and M, markers).

Article Snippet: After each cloning step, the forming arrays were digested with BamHI and NotI restriction enzymes (NEB) and analysed on 1% agarose gel electrophoresis using 100 bp DNA Ladder, Quick-Load 1 kb Extend DNA ladder or Low Range PFG Marker (NEB).

Techniques: Transformation Assay, Plasmid Preparation, Transfection, Marker

Journal: Scientific Reports

Article Title: ATP signaling in the integrative neural center of Aplysia californica

doi: 10.1038/s41598-021-84981-5

Figure Lengend Snippet: Distribution of AcP2X in the CNS of Aplysia and the effect of ATP on Aplysia F-cluster neurons. ( A , B ) AcP2X is expressed in neurons of the cerebral F-cluster (in situ hybridization). A pair of giant serotonergic feeding interneurons (MCC) are indicated by arrows. ( A ) The preparation embedded in a mounting media. ( B ) The cerebral ganglion was photographed in 100% ethanol. Scale 300 µm. ( C ) Current-clamp recording from F-cluster neurons in the intact CNS. Bath application of ATP (2.0 mM) caused an excitatory response with spiking activity (2–5 mV depolarization with a burst of the action potentials), and full recovery following washout (indicated by arrows) (n = 7, neurons). ( D ) Voltage-clamp recording from F-cluster neuron. Raw traces were recorded in response to 2.0 mM of ATP at three holding potentials (agonist application indicated by the line).

Article Snippet: The antisense probe was generated by digestion of the AcP2X plasmid with Not I (Cat#R0189s, New England Biolabs Inc.) then transcribed with T3 polymerase from the DIG RNA Labeling Kit (Cat#11175025910, Roche Diagnostics).

Techniques: In Situ Hybridization, Activity Assay

Journal: bioRxiv

Article Title: ATP signaling in the integrative neural center of Aplysia californica

doi: 10.1101/2020.11.20.392001

Figure Lengend Snippet: Distribution of AcP2X in the CNS of Aplysia and the effect of ATP on Aplysia F-cluster neurons. A and B : AcP2X is expressed in neurons of the cerebral F-cluster ( in situ hybridization). A pair of giant serotonergic feeding interneurons (MCC) are indicated by arrows. A . The preparation embedded in a mounting media. B . The cerebral ganglion was photographed in 100% ethanol. Scale: 300 μm. C . Current-clamp recording from F-cluster neurons in the intact CNS. Bath application of ATP (2.0 mM) caused an excitatory response with spiking activity (2-5 mV depolarization with a burst of the action potentials), and full recovery following washout (indicated by arrows). D . Voltage-clamp recording from F-cluster neuron. Raw traces recorded in response to 2.0 mM of ATP at three holding potentials (agonist application indicated by the line).

Article Snippet: The antisense probe was generated by digestion of the AcP2X plasmid with Not I (Cat#R0189s, New England Biolabs Inc.) then transcribed with T3 polymerase from the DIG RNA Labeling Kit (Cat#11175025910, Roche Diagnostics).

Techniques: In Situ Hybridization, Activity Assay