Journal:

Article Title: Role for Conserved Residues of Sindbis Virus Nonstructural Protein 2 Methyltransferase-Like Domain in Regulation of Minus-Strand Synthesis and Development of Cytopathic Infection ▿ †

doi: 10.1128/JVI.00224-08

Figure Lengend Snippet: (A) Schematic for the functional organization of nsP2 in the genus Alphavirus. The temperature-sensitive mutations (ts) that have been previously mapped to nsP2 helicase, protease, and the MTase-like domain are indicated. Mutations ts14, ts16, ts21, and ts19 are located in the helicase domain (62), and mutations ts18, ts17, ts133, and ts24 map to the protease domain (30). SFV ts4 is another mutation mapped to the extreme C terminus of nsP2 in SFV (71). (B) Location of the SINV nsP2 MTase-like domain residues chosen for mutagenesis on the VEEV nsP2pro structure. The N-terminal protease domain (residues 468 to 603) is shown in gray. The C-terminal MTase-like domain (605 to 787) is depicted in green. The mutated residues are labeled with VEEV numbering and SINV numbering in parentheses. The residues that were mutated in SINV are labeled with the VEEV numbering; e.g., SINV R615 is shown as VEEV R604 (Table ​(Table1).1). The residues are colored based on the plaque phenotype of the corresponding alanine mutants in SINV. Small plaque, blue; medium plaque, cyan. The ts lethal mutants are in magenta. The protease catalytic dyad and the residue that corresponds to the previously reported (23) noncytopathic mutation in SINV (SINV P726/VEEV P713), SIN/G, are indicated in orange.

Article Snippet: After transfer, the nitrocellulose membranes were processed by rabbit anti-nsP2 antibodies and an Alexa-Fluor 680-conjugated goat anti-rabbit secondary antibody (Molecular Probes).

Techniques: Functional Assay, Mutagenesis, Labeling

Journal:

Article Title: Role for Conserved Residues of Sindbis Virus Nonstructural Protein 2 Methyltransferase-Like Domain in Regulation of Minus-Strand Synthesis and Development of Cytopathic Infection ▿ †

doi: 10.1128/JVI.00224-08

Figure Lengend Snippet: Plaque phenotypes at permissive and nonpermissive temperatures of growth

Article Snippet: After transfer, the nitrocellulose membranes were processed by rabbit anti-nsP2 antibodies and an Alexa-Fluor 680-conjugated goat anti-rabbit secondary antibody (Molecular Probes).

Techniques: Mutagenesis

Journal:

Article Title: Role for Conserved Residues of Sindbis Virus Nonstructural Protein 2 Methyltransferase-Like Domain in Regulation of Minus-Strand Synthesis and Development of Cytopathic Infection ▿ †

doi: 10.1128/JVI.00224-08

Figure Lengend Snippet: Analysis of growth and viability for nsP2 MTase-like domain mutants. BHK-15 cells were infected with wild-type (SINV) or mutant virus at the same MOIs. The lethal mutants R615A and R699A, as well as the small-plaque mutants K733A and R751A, were compared with wild-type virus for relative growth rates. At the indicated times, medium was replaced, virus titers were determined by a standard plaque assay, and incubation was carried out at 30°C for 48 h. R699A did not show any detectable virus release.

Article Snippet: After transfer, the nitrocellulose membranes were processed by rabbit anti-nsP2 antibodies and an Alexa-Fluor 680-conjugated goat anti-rabbit secondary antibody (Molecular Probes).

Techniques: Infection, Mutagenesis, Plaque Assay, Incubation

Journal:

Article Title: Role for Conserved Residues of Sindbis Virus Nonstructural Protein 2 Methyltransferase-Like Domain in Regulation of Minus-Strand Synthesis and Development of Cytopathic Infection ▿ †

doi: 10.1128/JVI.00224-08

Figure Lengend Snippet: Phenotypic characteristics of MTase-like domain mutants

Article Snippet: After transfer, the nitrocellulose membranes were processed by rabbit anti-nsP2 antibodies and an Alexa-Fluor 680-conjugated goat anti-rabbit secondary antibody (Molecular Probes).

Techniques: In Vitro, In Vivo

Journal:

Article Title: Role for Conserved Residues of Sindbis Virus Nonstructural Protein 2 Methyltransferase-Like Domain in Regulation of Minus-Strand Synthesis and Development of Cytopathic Infection ▿ †

doi: 10.1128/JVI.00224-08

Figure Lengend Snippet: Analysis of protein synthesis and processing. (A) In vitro transcription and translation of nsP2 lethal (R615A, R699A, and K752A) and small-plaque mutants (K733A and R751A) using the rabbit reticulocyte lysate. Translation products were separated by SDS-PAGE and visualized by autoradiography. Locations of the proteins are indicated on the right. M indicates molecular mass (kDa). (B) Synthesis and processing of nsP2 in virus-infected cells. BHK cells in six-well plates were infected with wild-type (SINV) or mutant virus at an MOI of 5 and incubated at 30°C (I) or 37°C (II). At 12 h postinfection, cells were harvested and equal amounts of proteins were separated by SDS-10% PAGE. After transfer, the nitrocellulose membranes were processed by rabbit anti-nsP2 antibodies and an Alexa-Fluor 680-conjugated goat anti-rabbit secondary antibody (Molecular Probes). The K752A lane represents extracts obtained 12 h postelectroporation with Toto64 RNA carrying the mutation, which was tested previously along with mock and wild-type controls in a separate experiment.

Article Snippet: After transfer, the nitrocellulose membranes were processed by rabbit anti-nsP2 antibodies and an Alexa-Fluor 680-conjugated goat anti-rabbit secondary antibody (Molecular Probes).

Techniques: In Vitro, SDS Page, Autoradiography, Infection, Mutagenesis, Incubation

Journal:

Article Title: Role for Conserved Residues of Sindbis Virus Nonstructural Protein 2 Methyltransferase-Like Domain in Regulation of Minus-Strand Synthesis and Development of Cytopathic Infection ▿ †

doi: 10.1128/JVI.00224-08

Figure Lengend Snippet: Phenotypic characteristics of nsP2 interdomain loop mutants a

Article Snippet: After transfer, the nitrocellulose membranes were processed by rabbit anti-nsP2 antibodies and an Alexa-Fluor 680-conjugated goat anti-rabbit secondary antibody (Molecular Probes).

Techniques: In Vivo

Journal: Advanced Science

Article Title: mRNA‐Lipid Nanoparticle‐Mediated Restoration of PTPN14 Exhibits Antitumor Effects by Overcoming Anoikis Resistance in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309988

Figure Lengend Snippet: BCAR3 was identified as a substrate of PTPN14. A) The results of IP‐MS, following CompPASS analysis, yielded high‐confidence candidate PTPN14‐interacting proteins. B) Exogenous IP‐Western Blot analysis validated the interaction of PTPN14 and BCAR3. Three independent experiments were performed. C) Endogenous IP‐Western Blot analysis validated the interaction of PTPN14 and BCAR3. D) BCAR3 was knockdown in control or PTPN14‐KO MDA‐MB‐231 cells and cell viability under ULA conditions was assessed every 24 h for 5 days using the CCK8 assay (left); validation of protein expression levels in each cell group through Western Blotting (right). Three independent experiments were performed. E) After the knockdown of BCAR3 in PTPN14‐KO MDA‐MB‐231 cells, the phosphorylation levels of AKT and ERK were assessed. Three independent experiments were performed. F) Tyrosine phosphorylated protein IP‐Western Blot analysis was conducted in both control MDA‐MB‐231 cells and PTPN14‐KO MDA‐MB‐231 cells. Three independent experiments were performed. G) Tyrosine phosphorylated protein IP‐Western Blot analysis was conducted in both PTPN14‐HA MDA‐MB‐231 cells and PTPN14‐D1079A‐HA MDA‐MB‐231 cells.

Article Snippet: The following antibodies were used for Western Blot analysis: Cell Signaling Technology: PTPN14 (13808S, 1:1000 dilution), Cleaved PARP (5625S, 1:1000 dilution), Cleaved Caspase‐3 (9661S, 1:1000 dilution), p‐AKT (Ser473) (4060S, 1:2000 dilution), AKT (pan) (4691S, 1:1000 dilution), p‐ERK1/2 (4370S, 1:2000 dilution), ERK1/2 (4695S, 1:1000 dilution), BCAR3 (24032S, 1:1000 dilution), and HA‐Tag (3724S, 1:1000 dilution); Proteintech: GAPDH (60004‐1‐Ig, 1:50000 dilution), β‐actin (66009‐1‐Ig, 1:20000 dilution) and GFP tag (50430‐2‐AP, 1:1000 dilution); ABclonal: active + pro Caspase‐3 (A19654, 1:1000 dilution).

Techniques: Western Blot, Knockdown, Control, CCK-8 Assay, Expressing