Journal: The Journal of Biological Chemistry

Article Title: Isolation and structure elucidation of Dm -CVNH, a new cyanovirin-N homolog with activity against SARS-CoV-2 and HIV-1

doi: 10.1016/j.jbc.2025.108319

Figure Lengend Snippet: Binding isotherms and thermodynamic parameters of binding for Dm -CVNH with recombinant WH-1 and Omicron SARS-CoV-2 (HEK293-produced) Spike proteins. Isothermal titration calorimetry isotherms showing exothermic heats of binding between Dm -CVNH and WH-1 ( A ) and Omicron ( B ) Spike proteins, ( C ) the deconvoluted thermodynamic parameters of binding (enthalpy, entropy, free energy, binding affinity, and stoichiometry).

Article Snippet: PsV were prepared by transiently transfecting HEK293 T/17 cells (ATCC) with these three plasmids using Lipofectamine 3000 (Invitrogen) per manufacturer’s protocols.

Techniques: Binding Assay, Recombinant, Produced, Isothermal Titration Calorimetry

Journal: Journal of Biological Chemistry

Article Title: Protein Phosphatase 4 Is Involved in Tumor Necrosis Factor-α-induced Activation of c-Jun N-terminal Kinase

doi: 10.1074/jbc.m107014200

Figure Lengend Snippet: FIG. 1. Characterization of a PP4- specific antibody, Ab104. A, the anti- PP4 antibody, Ab104, specifically recog- nizes PP4, but not PP2A and PP6. HEK293T cells were transfected with 2 g of empty vector (lanes 1), 2 g of Flag- PP4 (lanes 2), 2 g of Flag-PP2A (lanes 3), or 2 g of Flag-PP6 (lanes 4). Cells were harvested 36 h after transfection and sub- jected to SDS-PAGE. Western blotting was performed with 1 g/ml Ab104. The experiments were repeated four times with similar results. B, PP4 co-purifies with centrosomes. Centrosomes were pre- pared from 6 107 HeLa cells and puri- fied on a discontinuous sucrose gradient. 10% of protein recovered from each frac- tion and 5 g of HeLa whole cell lysate (W) were Western-blotted for the presence of PP4 (Ab104) and subcellular compart- ment markers: -tubulin (centrosome), al- dolase (cytosol), lamin B1 (nucleus), GRP78 (endoplasmic reticulum), gol- gin-97 (Golgi), and Bcl-XL (mitochondria). C, PP4 is a component of the centrosome. HeLa cells were grown on polylysine- coated coverslips, extracted in 0.5% Tri- ton X-100 for 2 min, and fixed in 4% ul- trapure formaldehyde. Fixed cells were incubated with DAPI DNA stain (DAPI; blue), human autoimmune serum 4171 (PCM; red), and the peptide-purified anti- PP4 antibody Ab104 (PP4; green; panels a–d) or normal preimmune serum from the same rabbit used to generate Ab104, before immunization with peptide (n.s.; green; panels e–h). Panels PCM, PP4, and DAPI were merged (merged; panel d), to identify areas of colocalization of PP4 and PCM staining (yellow). Arrows indicate position of centrosomes. The experiments were repeated at least three times with similar results.

Article Snippet: Cells and Transfection—Human HeLa cells, human embryonic kidney 293T (HEK293T) and 293 (HEK293) cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and 100 units/ml streptomycin/penicillin at 37 °C in a humidified atmosphere of 5% CO2.

Techniques: Transfection, Plasmid Preparation, SDS Page, Western Blot, Incubation, Staining, Purification

Journal: Journal of Biological Chemistry

Article Title: Protein Phosphatase 4 Is Involved in Tumor Necrosis Factor-α-induced Activation of c-Jun N-terminal Kinase

doi: 10.1074/jbc.m107014200

Figure Lengend Snippet: FIG. 2. TNF- activates both PP4 and JNK in HEK293T cells. A, establishment of PP4 phosphatase assays. 800 g of HEK293 cell lysate was immunoprecipitated with either anti-PP4 antibody (Ab104) or pro- tein A bead alone. The immunoprecipitates were washed and incubated with assay buffer and KTpIRR peptide at 30 °C for various times, from 0 to 120 min, as indicated (upper panel). 200, 400, 600, or 800 g of HEK293 cell lysate was immunoprecipitated with either anti-PP4 an- tibody (Ab104) or beads alone. The immunoprecipitates were washed and incubated with assay buffer and KTpIRR peptide at 30 °C for 30 min (lower panel). The phosphatase assays were read at 650 nm. The readings are the average and standard deviation of three separate immunoprecipitations (PP4) or two separate immunoprecipitations (beads). B, TNF- activates PP4 phosphatase activity. HEK293T cells were seeded at a density of 3.5 106 cells/100-mm dish. After 24 h, the cells were treated with TNF- (10 ng/ml) for various times as indicated. PP4 was immunoprecipitated with an anti-PP4 antibody (Ab104). The PP4 phosphatase activity was determined by using a synthetic peptide, KTpIRR, as a substrate (upper panel). The amounts of PP4 immuno- precipitated were monitored by Western blotting using an anti-PP4 antibody (Ab6101; lower panel). The experiments were repeated at least three times with similar results. C, TNF- activates JNK kinase activ- ity. HEK293T cells were seeded at a density of 3.5 106 cells/100-mm dish. After 24 h, the cells were treated with TNF- (10 ng/ml) for various times as indicated. JNK was immunoprecipitated with an anti- JNK antibody (Ab101). The JNK phosphatase activity was determined by using GST-c-Jun-(1–79) as a substrate. The experiments were re- peated three times with similar results.

Article Snippet: Cells and Transfection—Human HeLa cells, human embryonic kidney 293T (HEK293T) and 293 (HEK293) cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and 100 units/ml streptomycin/penicillin at 37 °C in a humidified atmosphere of 5% CO2.

Techniques: Immunoprecipitation, Incubation, Standard Deviation, Activity Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Protein Phosphatase 4 Is Involved in Tumor Necrosis Factor-α-induced Activation of c-Jun N-terminal Kinase

doi: 10.1074/jbc.m107014200

Figure Lengend Snippet: FIG. 3. TNF- induces serine and threonine phosphorylation of PP4. A, TNF- induces PP4 phosphorylation. HEK293T cells (1 106 cells in 100-mm dish) were transfected with 5 g of Flag-PP4. After 40 h, the cells were labeled in phosphate-free DMEM supplemented with 5% of dialyzed serum and 100 Ci/ml [32P]orthophosphate for 4 h at 37 °C and treated with TNF- (10 ng/ml) for the period of time indicated. Flag-PP4 was immunoprecipitated with an anti-Flag anti- body (M2) and subjected to SDS-PAGE. The separated proteins were transferred to PVDF, and autoradiography was performed. B, TNF-- induced phosphorylation occurs on serine and threonine residues of PP4. The corresponding PP4 bands were cut from the PVDF membrane (A) and subjected to phosphoamino acid analysis. The experiments were repeated at least two times with similar results.

Article Snippet: Cells and Transfection—Human HeLa cells, human embryonic kidney 293T (HEK293T) and 293 (HEK293) cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and 100 units/ml streptomycin/penicillin at 37 °C in a humidified atmosphere of 5% CO2.

Techniques: Phospho-proteomics, Transfection, Labeling, Immunoprecipitation, SDS Page, Autoradiography, Membrane, Phosphoamino Acid Analysis

Journal: Journal of Biological Chemistry

Article Title: Protein Phosphatase 4 Is Involved in Tumor Necrosis Factor-α-induced Activation of c-Jun N-terminal Kinase

doi: 10.1074/jbc.m107014200

Figure Lengend Snippet: FIG. 4. JNK activation by TNF- is blocked by a phosphatase-dead PP4 mutant, PP4-RL. A, PP4-RL blocks TNF--induced JNK activation. HEK293T cells (1.5 105 cells in 35-mm wells) were transfected with HA-JNK (0.1 g) alone or HA-JNK plus PP4-RL (2g). Empty vector was used to normalize the amount of transfected DNA. 36 h after transfection, the cells were treated with TNF- (10 ng/ml) for 10 min. Cell lysates were prepared, HA-JNK1 was immunoprecipitated with an anti-HA antibody (12CA5), and immunocomplex kinase assays were performed using GST-c-Jun-(1–79) as a substrate (top panel). Expression levels of HA-JNK and HA-PP4-RL were monitored by immunoblotting using an anti-HA antibody (12CA5, lower panel). The experiments were repeated three times with similar results. B, PP4-RL is a phosphatase-dead mutant. HEK293T cells (1.0 106 cells in 100-mm dishes) were transfected with 10 g of either PP4 or HA-PP4-RL. The cells were collected 48 h after transfection. PP4 and HA-PP4-RL were immunoprecipitated with an anti-PP4 (Ab104) and an anti-HA (12CA5) antibody, respectively. The immunoprecipitates were then subjected to phosphatase assays (upper panel). The amounts of immunoprecipitated PP4 and HA-PP4-RL were monitored by Western blotting using an anti-PP4 antibody (Ab6101; lower panel). The experiments were repeated at least five times with similar results. C, TNF--induced JNK activation was inhibited in HEK293-PP4-RL cells. Parental HEK293 and HEK293-PP4-RL cells were seeded at a density of 4.5 106 cells in 100-mm dishes. After 24 h, the cells were treated with TNF- (10 ng/ml) for various times. Cell lysates were prepared, JNK1 was immunoprecipitated with an anti-JNK1 antibody (Ab101), and immunocomplex kinase assays were performed using GST-c-Jun-(1–79) as a substrate (left panel). Cell lysates from parental HEK293 cells and a HA-PP4-RL stably transfected clone, HEK293-PP4-RL, were subjected to SDS-PAGE and Western blotting with an anti-PP4 antibody (Ab104) or an anti-HA antibody (12CA5, right panel).

Article Snippet: Cells and Transfection—Human HeLa cells, human embryonic kidney 293T (HEK293T) and 293 (HEK293) cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and 100 units/ml streptomycin/penicillin at 37 °C in a humidified atmosphere of 5% CO2.

Techniques: Activation Assay, Mutagenesis, Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Western Blot, Stable Transfection, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Protein Phosphatase 4 Is Involved in Tumor Necrosis Factor-α-induced Activation of c-Jun N-terminal Kinase

doi: 10.1074/jbc.m107014200

Figure Lengend Snippet: FIG. 5. PP4 activates JNK in transient transfection assay. HEK293T cells (1.5 105 cells in 35-mm wells) were transfected with HA-JNK1 (0.1 g) alone or with 2 g of PP4, PP1, or Myc-M3/6. Empty vector was used to normalize the amount of transfected DNA. 36 h after transfection, the cell lysates were prepared. HA-JNK1 was immunopre- cipitated with an anti-HA antibody (12CA5), and immunocomplex ki- nase assays were performed using GST-c-Jun-(1–79) as a substrate. Expression levels of HA-JNK1, PP4, PP1, and Myc-M3/6 were moni- tored by immunoblotting using anti-HA (12CA5), anti-PP4 (Ab104), anti-PP1, and anti-Myc antibodies, respectively (bottom panels). The experiments were repeated at least 10 times with similar results.

Article Snippet: Cells and Transfection—Human HeLa cells, human embryonic kidney 293T (HEK293T) and 293 (HEK293) cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and 100 units/ml streptomycin/penicillin at 37 °C in a humidified atmosphere of 5% CO2.

Techniques: Transient Transfection Assay, Transfection, Plasmid Preparation, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Protein Phosphatase 4 Is Involved in Tumor Necrosis Factor-α-induced Activation of c-Jun N-terminal Kinase

doi: 10.1074/jbc.m107014200

Figure Lengend Snippet: FIG. 6. PP4 specifically activates JNK, but not p38 and ERK2. A, HEK293T cells (1.5 105 cells in 35-mm wells) were transfected with HA-JNK1 (0.5 g) alone or HA-JNK1 plus various amounts of PP4 as indicated. Empty vector was used to normalize the amount of trans- fected DNA. 36 h after transfection, the cell lysates were prepared. HA-JNK1 was immunoprecipitated with an anti-HA antibody (12CA5), and immunocomplex kinase assays were performed using GST-c-Jun- (1–79) as a substrate. Expression levels of HA-JNK1 and PP4 were monitored by immunoblotting using anti-HA (12CA5) and anti-PP4 antibodies, respectively (bottom panels). The experiments were re- peated at least 10 times with similar results. B, HEK293T cells (1.5 105 cells in 35-mm wells) were transfected with HA-p38 (1 g) alone, HA-p38 plus various amounts of PP4, or HA-p38 plus 2 g of HA- MKK6, as indicated. Empty vector was used to normalize the amount of transfected DNA. 36 h after transfection, the cell lysates were prepared. HA-p38 was precipitated with an anti-HA antibody (12CA5), and im- munocomplex kinase assays were performed using GST-ATF2-(1–96) as a substrate. Expression levels of HA-p38, HA-MKK6, and PP4 were monitored by immunoblotting using anti-HA (12CA5) and anti-PP4 antibodies, respectively (bottom panels). C, HEK293T cells (1.5 105

Article Snippet: Cells and Transfection—Human HeLa cells, human embryonic kidney 293T (HEK293T) and 293 (HEK293) cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and 100 units/ml streptomycin/penicillin at 37 °C in a humidified atmosphere of 5% CO2.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Protein Phosphatase 4 Is Involved in Tumor Necrosis Factor-α-induced Activation of c-Jun N-terminal Kinase

doi: 10.1074/jbc.m107014200

Figure Lengend Snippet: FIG. 7. PP4 does not interact with JNK in vitro. A, no PP4-JNK association was detected in vitro. HEK293 cells stably transfected with Flag-PP4 (10F1 clone) were seeded at 4 106 cells/100-mm dish and treated with TNF- (10 ng/ml) for 5 min the next day. 600 g of lysate from either TNF--treated or untreated 10F1 HEK293 cells was incu- bated with GST or GST-JNK fusion protein immobilized onto glutathi- one-agarose beads for 2 h at 4 °C. The PP4-JNK interaction was ana- lyzed by immunoblotting with an anti-Flag antibody (M2) to detect Flag-PP4 bound to GST-JNK after SDS-PAGE (upper panel). The GST and GST-JNK were monitored by immunoblotting with an anti-GST antibody (middle panel). The experiments were repeated three times with similar results. To assure Flag-PP4 was in a phosphorylated state, 10F1 HEK293 cells were labeled in the phosphate-free DMEM supple- mented with 5% of dialyzed serum and 100 Ci/ml [32P]orthophosphate for 4 h at 37 °C and treated with TNF- (10 ng/ml) for 5 min. Flag-PP4 was immunoprecipitated with an anti-Flag antibody (M2) and subjected to SDS-PAGE and autoradiography. The experiments were repeated two times with similar results. B, M3/6 associates with JNK in vitro. GST or GST-JNK fusion protein was immobilized on glutathione-aga- rose beads and incubated with 600 g of lysate from HEK293T cells transiently transfected with Myc-M3/6 for 2 h at 4 °C. The M3/6-JNK interaction was analyzed by immunoblotting with an anti-Myc antibody to detect Myc-M3/6 bound to GST-SAPK after SDS-PAGE (upper pan- el). The GST and GST-SAPK were monitored by immunoblotting with an anti-GST antibody (lower panel).

Article Snippet: Cells and Transfection—Human HeLa cells, human embryonic kidney 293T (HEK293T) and 293 (HEK293) cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and 100 units/ml streptomycin/penicillin at 37 °C in a humidified atmosphere of 5% CO2.

Techniques: In Vitro, Stable Transfection, Transfection, Western Blot, SDS Page, Labeling, Immunoprecipitation, Autoradiography, Incubation