Journal: Journal of Virology

Article Title: Measles Virus Infection Inactivates Cellular Protein Phosphatase 5 with Consequent Suppression of Sp1 and c-Myc Activities

doi: 10.1128/JVI.00825-15

Figure Lengend Snippet: Inactivation of PP5 correlates with the suppression of DNA-PK activity. (A) 293SLAM cells were infected with MeV for 24 h and then immunoprecipitated with anti-DNA-PKcs, anti-PP2A, anti-PP5, or anti-PP6c antibody. Phosphatase activity in immunocomplexes was determined by an in vitro phosphatase assay. The results are expressed as values relative to the value of the mock-treated control sample (upper panel). The protein level of each protein was detected by immunoblotting (lower panel). (B) Knockdown of PP5 by RNA interference. 293SLAM cells were transfected with siRNA targeting PP5. For controls, cells were either mock treated or transfected with a nonspecific mismatched control siRNA (ctrl). After 48 h, the cells were lysed and subjected to immunoblotting or EMSA, or they were radiolabeled with 32PO4 and immunoprecipitated with anti-Sp1 antibody. (C) The cell lysates in panel B were subjected to immunoblotting with anti-c-Myc and anti-PP5 antibodies. (D) Total RNA prepared from cells transfected with ctrl siRNA or siRNA targeting PP5 described in panel B were then subjected to qPCR. The expression level in PP5 knock down cells was represented as a value relative to the that of ctrl siRNA-transfected cells. The specific primer pairs used are indicated by numbered columns as follows: 1, RNA18S5; 2, HIST1H2AC; 3, CASP3; 4, JAK1; 5, MAPK6; 6, IRF1; 7, NDUFB4; 8, COX7C; 9, RPL23; 10, LAMA4; 11, HSPA1L; and 12, ATP5G3. The official full names of these genes are described in the supplemental material.

Article Snippet: The primary antibodies were purchased from the following manufacturers: anti-Sp1 (rabbit polyclonal; Santa Cruz Biotechnology), anti-DNA-PKcs (mouse monoclonal, clone 18-2; GeneTex), anti-PP2A catalytic subunit (mouse monoclonal, clone 46; BD Biosciences), anti-PP5 (goat polyclonal; Santa Cruz Biotechnology), anti-PP6c (rabbit polyclonal; GeneTex), anti-Ku86 (mouse monoclonal, clone S10B1; Calbiochem), anti-glyceraldehyde phosphate dehydrogenase (GAPDH) (mouse monoclonal, clone MAB374; Chemicon), and anti-c-Myc (mouse monoclonal, clone 9E10; Clontech).

Techniques: Activity Assay, Infection, Immunoprecipitation, In Vitro, Phosphatase Assay, Western Blot, Transfection, Expressing

Journal: Journal of Virology

Article Title: Measles Virus Infection Inactivates Cellular Protein Phosphatase 5 with Consequent Suppression of Sp1 and c-Myc Activities

doi: 10.1128/JVI.00825-15

Figure Lengend Snippet: Accumulation of intracellular viral nucleocapsid inactivates PP5. (A) HEK293 cells were transfected with expression plasmids encoding MeV-N, -P, and -L genes (N, P, and L) or control empty plasmid, together with phRG-B as an internal reference. The following day, the cells were transfected with mock or negative-strand minigenomic RNA (minigenome). The cells were harvested at 24, 36, and 48 h after RNA transfection, and their lysates were subjected to luciferase activity analysis (upper panel), immunoblotting for MeV-N (middle panel), and an in vitro phosphatase assay with anti PP5 antibody (lower panel), as described above. (B) 293SLAM cells were transfected with N, P, and L expression plasmids and then transfected with mock or minigenomic RNA the following day. At 48 h after RNA transfection, cell lysates were immunoprecipitated with anti-PP5 or anti-PP2A antibody. Phosphatase activity in the immunocomplexes was determined as described above. (C) Nuclear extracts prepared from cells under the same conditions as panel B were subjected to EMSA. (D) The cell lysates from panel B and mock-treated cell lysates were immunoblotted to detect c-Myc, MeV-N, and GAPDH.

Article Snippet: The primary antibodies were purchased from the following manufacturers: anti-Sp1 (rabbit polyclonal; Santa Cruz Biotechnology), anti-DNA-PKcs (mouse monoclonal, clone 18-2; GeneTex), anti-PP2A catalytic subunit (mouse monoclonal, clone 46; BD Biosciences), anti-PP5 (goat polyclonal; Santa Cruz Biotechnology), anti-PP6c (rabbit polyclonal; GeneTex), anti-Ku86 (mouse monoclonal, clone S10B1; Calbiochem), anti-glyceraldehyde phosphate dehydrogenase (GAPDH) (mouse monoclonal, clone MAB374; Chemicon), and anti-c-Myc (mouse monoclonal, clone 9E10; Clontech).

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, In Vitro, Phosphatase Assay, Immunoprecipitation

Journal: Journal of Virology

Article Title: Measles Virus Infection Inactivates Cellular Protein Phosphatase 5 with Consequent Suppression of Sp1 and c-Myc Activities

doi: 10.1128/JVI.00825-15

Figure Lengend Snippet: Model of the MeV-induced signal that triggers the comprehensive downregulation of cellular housekeeping gene expression. The intracellular accumulation of viral nucleocapsid causes the inactivation of PP5, autophosphorylation, and the resulting inactivation of DNA-PKcs and then the downstream reduction of Sp1 phosphorylation and degradation of c-Myc, both of which cause their inactivation and consequent downregulation of housekeeping genes in 293SLAM cells.

Article Snippet: The primary antibodies were purchased from the following manufacturers: anti-Sp1 (rabbit polyclonal; Santa Cruz Biotechnology), anti-DNA-PKcs (mouse monoclonal, clone 18-2; GeneTex), anti-PP2A catalytic subunit (mouse monoclonal, clone 46; BD Biosciences), anti-PP5 (goat polyclonal; Santa Cruz Biotechnology), anti-PP6c (rabbit polyclonal; GeneTex), anti-Ku86 (mouse monoclonal, clone S10B1; Calbiochem), anti-glyceraldehyde phosphate dehydrogenase (GAPDH) (mouse monoclonal, clone MAB374; Chemicon), and anti-c-Myc (mouse monoclonal, clone 9E10; Clontech).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Aberrant Expression and Secretion of Heat Shock Protein 90 in Patients with Bullous Pemphigoid

doi: 10.1371/journal.pone.0070496

Figure Lengend Snippet: Expression of ( a ) TGF-α, ( b ) protein phosphatase 5 (PP5) and ( c ) protein kinase A (PKA) was analyzed in serum (TGF-α) and peripheral blood mononuclear cells (PBMC) lysates (PP5 and PKA) of bullous pemphigoid (BP) patients and healthy controls by enzyme-linked immunosorbent assay and western blotting, respectively, and revealed no significant difference between both study groups. ( d ) Mean PP5 and PKA concentrations were expressed relative to the β-Actin level using densitometry measurements of immunoblots. Values are mean ± SEM of 3–9 BP patients and 3–6 controls.

Article Snippet: The membrane was blocked with 3% milk in PBS for 2 hours, followed by incubation with rabbit polyclonal antibodies against protein phosphatase 5 (PP5; 1∶100; Abcam), protein kinase A (PKA) αβ (1∶1000; Abcam), and β-Actin (1∶1000; Sigma) at RT for 2 hours.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot