Journal: Biochemical and Biophysical Research Communications

Article Title: The SARS-CoV S glycoprotein: expression and functional characterization

doi: 10.1016/j.bbrc.2003.11.054

Figure Lengend Snippet: Cell fusion mediated by the S glycoprotein. (A) Syncytium formation between 293T cells transfetced with pSecTag2B-S and pCDNA3-ACE2, respectively (right panel). There was no syncytium formation between 293T cells transfected with pSecTag2B-S and pCDNA3-ACE2-Ecto (left panel). (B) Cell fusion measured by a reporter gene-based assay. S glycoprotein expressed in both pCDNA3 and pSecTag2B vectors can be used in a β-gal reporter gene-based cell–cell fusion assay. A pCDNA3-based plasmid without S insert was used as plasmid control, and fusion between S-expressing cells with ACE2-ecto expressing cells was used as negative control.

Article Snippet: The fragment was cut with Bam HI and Apa I and cloned into corresponding sites of expression vector pSecTag2B (Invitrogen).

Techniques: Transfection, Reporter Gene Assay, Cell-Cell Fusion Assay, Plasmid Preparation, Expressing, Negative Control

Journal: Horticulture Research

Article Title: Transcriptomic analysis of interstock-induced dwarfism in Sweet Persimmon ( Diospyros kaki Thunb.)

doi: 10.1038/s41438-019-0133-7

Figure Lengend Snippet: a Fragment sizes predicted by ApaI digestion in ‘Nantong-xiaofangshi’, Diospyros lotus and ‘Kanshu’. b Polyacrylamide gel electrophoresis of restrictive digestion of different RT-PCR products. c Samples from an 82 cm tall grafted tree with ‘Kanshu’ as scion, ‘Nantong-xiaofangshi’ as interstock and Diospyros lotus as rootstock. d CAPS analysis on purified RT-PCR products of different tissues in ‘Kanshu’/‘Nantong-xiaofangshi’/ Diospyros lotus . e Comparison of total DkGA2ox1 / DLGA2ox1 / KSGA2ox1 transcripts in ‘Kanshu’/‘Nantong-xiaofangshi’/ Diospyros lotus . DkGA2ox1 , DLGA2ox1 and KSGA2ox1 are the sequences in ‘Nantong-xiaofangshi’, Diospyros lotus and ‘Kanshu’, respectively; N.x: ‘Nantong-xiaofangshi’ / Diospyros lotus , DL: Diospyros lotus , KS: ‘Kanshu’, M’: mixture of cDNA from N.x, DL and KS2, KS1: ‘Kanshu’ /‘Nantong-xiaofangshi’/ Diospyros lotus , KS2: ‘Kanshu’ / Diospyros lotus , GU1: graft union of Diospyros lotus and ‘Nantong-xiaofangshi’, IN: ‘Nantong-xiaofangshi’ interstock, GU2: graft union of ‘Nantong-xiaofangshi’ and ‘Kanshu’, 1-3: ‘Kanshu’ scion every 10 cm from the GU2, 4: young leaves at the top of the scion. M: DNA ladder marker 500. Scale bars = 5 cm. Note: double asterisk (**) indicates a significant difference ( P < 0.01). The purified PCR products were diluted to the same concentration for enzyme cutting

Article Snippet: Restriction enzyme cleavage sites of Diospyros lotus , N.x, and ‘Kanshu’ were constructed, and a specific ApaI cut site was identified (Thermo Scientific, Waltham, MA, USA).

Techniques: Polyacrylamide Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Purification, Comparison, Marker, Concentration Assay

Journal:

Article Title: IDENTIFICATION AND CHARACTERIZATION OF A NOVEL ENDOGENOUS MURINE PARKIN MUTATION

doi: 10.1111/j.1471-4159.2010.06605.x

Figure Lengend Snippet: Abolishment of the PRK8 and PRK28 antibody epitopes due to the E399Q human parkin mutant. A) pDEST15/parkin 380–465 and pDEST15/parkin 380–465 E399Q constructs were used to express the respective proteins in BL21 E. coli. Total protein lysates were extracted and equal amounts of protein (600 ng) were resolved on 13% polyacrylamide gels and analyzed by western blot with GST, PRK8, PRK28, and PRK109 antibodies. B) Full-length untagged wild-type and mutant (T240R and E399Q) human parkin cDNA cloned in the mammalian express vector pcDNA3.1 were used to express these proteins in mouse N2A neuroblastoma cells. Following transfection with the respective constructs, equal amounts (4 ug) of total protein lysates were resolved on 13% polyacrylamide gels and assessed by western blot for recognition of the indicated parkin antibodies. Additionally, the blots were probed with an actin antibody to confirm equal protein loading. The mobility of molecular mass markers is indicated on the left.

Article Snippet: Generation of human parkin constructs for expression in mammalian cells The full-length untagged wild-type (“WT”) human parkin cDNA was cloned into the XhoI and Apa I restriction sites on the pcDNA3.1(+) mammalian expression vector (Invitrogen).

Techniques: Mutagenesis, Construct, Western Blot, Clone Assay, Plasmid Preparation, Transfection

Journal:

Article Title: IDENTIFICATION AND CHARACTERIZATION OF A NOVEL ENDOGENOUS MURINE PARKIN MUTATION

doi: 10.1111/j.1471-4159.2010.06605.x

Figure Lengend Snippet: E399Q mutant parkin is functionally impaired. HEK293T cells were co-transfected either with mock pcDNA3.1 vector “(−)” or full length WT, T240R, or E399Q human parkin pcDNA 3.1 constructs as well as the pcDNA3.1 myc-tagged full-length synphilin-1 construct. The co-transfection experiments were performed using a parkin to synphilin-1 cDNA ratio of 4:1. At 24 hours post transfection, (A and B) cells were incubated for 16 hours with fresh DMEM or with DMEM containing 5uM Omuralide. Cells were then harvested and total protein lysates were extracted. Extracts were resolved by 10 % polyacrylamide gels and immunoblotted with the monoclonal antibodies anti-c-Myc clone 9E10 to assess synphilin-1 levels, PRK109 antibody to confirm parkin expression, and anti-actin antibody to ensure equal protein loading. All experiments were performed in triplicate and repeated at least 3 times. The mobility of molecular mass markers is indicated on the left. The levels of synphilin-1 were quantified as described in “Materials and Methods” and the graph in B depicts the percentage of synphilin-1 protein standardized to that in the mock vector samples. The error bars indicate standard deviation between replicate samples (n=4). C) Cells were pulsed with 35S-methionine for 1 hour and chased for 0, 1, 3, or 6 hours. The inset shows representative pulse-chase experiments. Experiments were conducted in triplicates. The results are plotted as percentage of protein over time standardized to the 0 hrs time point. The error bars show standard deviation (n=3).

Article Snippet: Generation of human parkin constructs for expression in mammalian cells The full-length untagged wild-type (“WT”) human parkin cDNA was cloned into the XhoI and Apa I restriction sites on the pcDNA3.1(+) mammalian expression vector (Invitrogen).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Construct, Cotransfection, Incubation, Expressing, Standard Deviation, Pulse Chase

Journal:

Article Title: IDENTIFICATION AND CHARACTERIZATION OF A NOVEL ENDOGENOUS MURINE PARKIN MUTATION

doi: 10.1111/j.1471-4159.2010.06605.x

Figure Lengend Snippet: E399Q mutant parkin can directly interact with synphilin-1. HEK293T cells were transiently co-transfected with myc-tagged synphilin-1 and pcDNA3.1 mock vector (“none”) or with untagged full-length WT, T240R, or E399Q human parkin pcDNA3.1 constructs. Cells were harvested and soluble protein lysates were extracted (“Input”). The cell lysates were then immunoprecipitated using anti-c-Myc polyclonal antibody (“IP”). Equal amounts of the input, IP, and unbound supernatant (Unbound) fractions were resolved by SDS-PAGE and analyzed by western blot (“WB”) with anti-c-Myc monoclonal antibody 9E10 or with anti-parkin antibody PRK109. Experiments were repeated at least 3 times. The mobility of molecular mass markers is indicated on the left.

Article Snippet: Generation of human parkin constructs for expression in mammalian cells The full-length untagged wild-type (“WT”) human parkin cDNA was cloned into the XhoI and Apa I restriction sites on the pcDNA3.1(+) mammalian expression vector (Invitrogen).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Construct, Immunoprecipitation, SDS Page, Western Blot

Journal:

Article Title: IDENTIFICATION AND CHARACTERIZATION OF A NOVEL ENDOGENOUS MURINE PARKIN MUTATION

doi: 10.1111/j.1471-4159.2010.06605.x

Figure Lengend Snippet: The E399Q Parkin mutant shows reduced binding to UbcH7 and UbcH8. HEK293T cells were co-transfected with pRK5-HA-UbcH7 or pRK5-HA-UbcH8 and either pcDNA3.1 mock vector (“mock”) or with untagged full-length WT, T240R, or E399Q human parkin pcDNA3.1 constructs. Cells were harvested and soluble protein lysates were extracted (“Input”). The cell lysates were then immunoprecipitated using the anti-HA polyclonal antibody, HA.11 (“IP”). Input and IP fractions were resolved by SDS-PAGE and analyzed by immunoblot with the monoclonal antibodies PRK109 and anti-HA (clone 12CA5). The PRK109 blots are shown at 5 second and 20 second exposure times to highlight the differences in signal intensity reflected by the binding of parkin with UbcH7 versus that with UbcH8.

Article Snippet: Generation of human parkin constructs for expression in mammalian cells The full-length untagged wild-type (“WT”) human parkin cDNA was cloned into the XhoI and Apa I restriction sites on the pcDNA3.1(+) mammalian expression vector (Invitrogen).

Techniques: Mutagenesis, Binding Assay, Transfection, Plasmid Preparation, Construct, Immunoprecipitation, SDS Page, Western Blot