Journal: International Journal of Molecular Sciences

Article Title: Jurkat T-Cell Antigen-Independent Elimination of PMA-Activated Neuroblastoma Cells Is Triggered by CCL2/CCR2, Depends Upon Lipid Raft LFA1/ICAM1 Immune Synapses, Is Mediated by m-TRAIL and Is Augmented by the TrkAIII Oncoprotein

doi: 10.3390/ijms27041970

Figure Lengend Snippet: Line graph demonstrating significant reductions in the percentage of PMA-activated TrkAIII-SH-SY5Y cells eliminated by Jurkat (Control) in the presence of entrectinib (1 μM), NCS87877 (1 μM), PP1 (1 μM), LY294002 (25 μM), capivasertib (50 μM) or PD98059 (10 μM) at 6, 12 and 24 h in duplicate experiments, each performed in duplicate (* p < 0.0001).

Article Snippet: UCF-101, z-VAD-fmk, z-IETD-fmk, z-DEVD-fmk, INCB3284, Capivasertib, NSC-87877, BIRT377, PP1, MET12, soluble recombinant TRAIL (s-TRAIL), recombinant human ICAM-1 (rICAM-1), IT1t-dihydrochloride, Bindarit and recombinant human osteoprotegerin were from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control

Journal: Molecular and Cellular Biology

Article Title: Growth Arrest and DNA Damage-Inducible Protein GADD34 Assembles a Novel Signaling Complex Containing Protein Phosphatase 1 and Inhibitor 1

doi: 10.1128/mcb.21.20.6841-6850.2001

Figure Lengend Snippet: FIG. 1. Identification of GADD34 as a protein interacting with PP1 and I-1. Yeast strains containing pACTII-GADD34 and either pASI- I-1, pAS-GLC7, or control vector pAS-Gi12 were grown on nonselec- tive medium lacking Trp and Leu and selective medium lacking Trp, Leu, Ade, and His as described in the text. The figure shows the strength of the protein-protein interactions as estimated by a liquid -galactosidase assay, with standard errors.

Article Snippet: Anti-GAL4 and anti-GADD34 antibodies were purchased from Santa Cruz, and anti-PP1 antibody was obtained from Transduction Laboratories.

Techniques: Control, Plasmid Preparation, Protein-Protein interactions

Journal: Molecular and Cellular Biology

Article Title: Growth Arrest and DNA Damage-Inducible Protein GADD34 Assembles a Novel Signaling Complex Containing Protein Phosphatase 1 and Inhibitor 1

doi: 10.1128/mcb.21.20.6841-6850.2001

Figure Lengend Snippet: FIG. 2. I-1 associates directly with GADD34. (A) GADD34-related proteins, shown schematically with regions of structural and functional homology. These proteins also show structural homology with two viral proteins, HSV-1 ICP34.5 and ASV NL-S, specifically in the C-terminal domains containing putative KIXF PP1-binding motifs. (B) Association of recombinant His-GADD34(230–674) with thiophosphorylated (thio-I-1) and unphosphorylated I-1 covalently linked to Sepharose. Controls included albumin and GST-GM(1–240), the PP1-binding fragment of the skeletal muscle glycogen-targeting subunit, which were also covalently linked to Sepharose. Input shows that almost all GADD34 was bound by immobilized I-1. GADD34 binding was visualized using both Coomassie blue protein stain and Western immunoblotting with anti-GADD34 antibody.

Article Snippet: Anti-GAL4 and anti-GADD34 antibodies were purchased from Santa Cruz, and anti-PP1 antibody was obtained from Transduction Laboratories.

Techniques: Functional Assay, Binding Assay, Recombinant, Staining, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Growth Arrest and DNA Damage-Inducible Protein GADD34 Assembles a Novel Signaling Complex Containing Protein Phosphatase 1 and Inhibitor 1

doi: 10.1128/mcb.21.20.6841-6850.2001

Figure Lengend Snippet: FIG. 3. Mapping GADD34 binding to I-1. (A) Schematic of I-1 structure, with the N-terminal 54 residues (cross-hatched) previously shown to bind PP1 and the PKA-phosphorylated threonine (T35P) required for PP1 inhibition marked. Polypeptides representing C-ter- minal truncations of I-1 were expressed in bacteria as hexahistidine- tagged proteins and purified by affinity chromatography on Ni-NTA- agarose. Purity of I-1 proteins is shown by SDS-PAGE on a 10% (wt/vol) polyacrylamide gel stained with Coomassie blue. The I-1 pro- teins were covalently linked to CNBr-activated Sepharose and used in pulldowns of recombinant His-GADD34(230–674) (B). Pulldowns of PP1 catalytic subunits purified from rabbit skeletal muscle were also undertaken using the immobilized unphosphorylated (B) and phos- phorylated I-1 (C). The results are compared with thiophosphorylated I-1 immobilized to Sepharose. I-1-bound proteins were analyzed by SDS-PAGE and Western immunoblotting (WB) with anti-GADD34 and anti-PP1 antibodies.

Article Snippet: Anti-GAL4 and anti-GADD34 antibodies were purchased from Santa Cruz, and anti-PP1 antibody was obtained from Transduction Laboratories.

Techniques: Binding Assay, Inhibition, Bacteria, Chromatography, SDS Page, Staining, Recombinant, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Growth Arrest and DNA Damage-Inducible Protein GADD34 Assembles a Novel Signaling Complex Containing Protein Phosphatase 1 and Inhibitor 1

doi: 10.1128/mcb.21.20.6841-6850.2001

Figure Lengend Snippet: FIG. 4. Mapping PP1 and I-1 binding to GADD34. FLAG-tagged GADD34 proteins (schematically shown in panel A with FLAG tag and PP1-binding sites highlighted) were expressed in HEK293T cells. Immunoblotting of total cell extracts with anti-FLAG antibody estab- lished relative expression of the GADD34 polypeptides in cells (B). HEK293T cell extracts were incubated with microcystin-LR-Sepharose (labeled Mcyst pulldown) to isolate cellular PP1/GADD34 complexes, and GADD34 was detected by Western immunoblotting (WB) (C). I-1-Sepharose was used to isolate GADD34 from HEK293T cell ex- tracts, and the bound proteins in the “I-1 pulldown” were analyzed by immunoblotting with the anti-HA antibody (D).

Article Snippet: Anti-GAL4 and anti-GADD34 antibodies were purchased from Santa Cruz, and anti-PP1 antibody was obtained from Transduction Laboratories.

Techniques: Binding Assay, FLAG-tag, Western Blot, Expressing, Incubation, Labeling

Journal: Molecular and Cellular Biology

Article Title: Growth Arrest and DNA Damage-Inducible Protein GADD34 Assembles a Novel Signaling Complex Containing Protein Phosphatase 1 and Inhibitor 1

doi: 10.1128/mcb.21.20.6841-6850.2001

Figure Lengend Snippet: FIG. 5. Modulation of PP1 activity by GADD34. (A) PP1 catalytic subunit isolated from rabbit skeletal muscle was assayed using 32P- labeled phosphorylase a and eIF-2 as substrates in the presence of increasing concentrations of recombinant His-tagged GADD34(230– 674). A representative assay from three independent experiments that varied by less than 5% is shown. (B) Effects of increasing concentra- tions of recombinant human GADD34(230–674) and human GST- GM(1–240) on the in vitro dephosphorylation of eIF-2 by skeletal muscle PP1 catalytic subunit are shown, with standard error bars. The results represent the sum of three independent experiments carried out in duplicate.

Article Snippet: Anti-GAL4 and anti-GADD34 antibodies were purchased from Santa Cruz, and anti-PP1 antibody was obtained from Transduction Laboratories.

Techniques: Activity Assay, Isolation, Labeling, Recombinant, In Vitro, De-Phosphorylation Assay

Journal: Molecular and Cellular Biology

Article Title: Growth Arrest and DNA Damage-Inducible Protein GADD34 Assembles a Novel Signaling Complex Containing Protein Phosphatase 1 and Inhibitor 1

doi: 10.1128/mcb.21.20.6841-6850.2001

Figure Lengend Snippet: FIG. 6. Effect of GADD34 on PP1 inhibition by I-1. The dose- dependent inhibition of phosphorylase phosphatase activity of the skeletal muscle PP1 catalytic subunit was analyzed in the presence (triangles) and absence (diamonds) of 50 nM recombinant GST- GM(1–240) (A) and 30 nM His-GADD34(230–674) (B). Representa- tive curves from three independent experiments that varied by less than 5% are shown. (C) Inhibition of eIF-2 phosphatase activity of the PP1 catalytic subunit by I-1 in the absence and presence of 50 nM His-GADD34(230–674). Results are the sum of three different exper- iments carried out in duplicate and are shown with standard error bars.

Article Snippet: Anti-GAL4 and anti-GADD34 antibodies were purchased from Santa Cruz, and anti-PP1 antibody was obtained from Transduction Laboratories.

Techniques: Inhibition, Activity Assay, Recombinant

Journal: Molecular and Cellular Biology

Article Title: Growth Arrest and DNA Damage-Inducible Protein GADD34 Assembles a Novel Signaling Complex Containing Protein Phosphatase 1 and Inhibitor 1

doi: 10.1128/mcb.21.20.6841-6850.2001

Figure Lengend Snippet: FIG. 7. Physiological regulation of PP1/GADD34 and I-1/ GADD34 complexes. (A) Comparison of total amounts of PP1 cata- lytic subunit, I-1, eIF-2, and GADD34 in extracts from brains of active and hibernating squirrel assessed by Western immunoblotting with the appropriate antibodies. (B) Levels of I-1 phosphorylated on threonine-35 and eIF-2 phosphorylated on serine-51 in brain extracts by immunoblotting with the relevant phosphospecific antibodies. (C) I-1 was immunoprecipitated from squirrel brain extracts using a poly- clonal anti-human I-1 antibody. The immunoprecipitates were sub- jected to SDS-PAGE and blotted with an anti-GADD34 antibody. (D) Microcystin-LR-Sepharose was used to isolate PP1 complexes from squirrel brain extracts. The presence of PP1 and GADD34 in micro- cystin-LR-bound complexes from brain extracts from active (left lanes) and hibernating (right lanes) ground squirrels was analyzed by immu- noblotting with anti-PP1 and anti-GADD34 antibodies, respectively.

Article Snippet: Anti-GAL4 and anti-GADD34 antibodies were purchased from Santa Cruz, and anti-PP1 antibody was obtained from Transduction Laboratories.

Techniques: Comparison, Western Blot, Immunoprecipitation, SDS Page

Journal: Journal of Translational Medicine

Article Title: Young and undamaged recombinant albumin alleviates T2DM by improving hepatic glycolysis through EGFR and protecting islet β cells in mice

doi: 10.1186/s12967-023-03957-3

Figure Lengend Snippet: rMSA activated SRC and reduced the interaction of CAV1 between EGFR to increase EGFR activation. A Western blot showing the protein and phosphorylation levels of IGF-1R/IR and AKT in the AML12 hepatocytes treated with vehicle or rMSA (75, 600 μM), with or without insulin (1, 10 nM) for 5 min. B Western blot showing the protein and phosphorylation levels of IGF-1R/IR in the AML12 hepatocytes treated with vehicle or rMSA (600 μM), with or without insulin (10 nM) at different time points. C Western blot showing the protein and phosphorylation levels of SRC in the AML12 hepatocytes treated with rMSA (600 μM) at different time points. D Western blot showing the protein and phosphorylation levels of EGFR, SRC, and AKT in the AML12 hepatocytes treated with vehicle or rMSA (600 μM) in the absence or presence of 20 μM PP1 or 2 μM Dasatinib for 2 h. E Western blot showing the protein and phosphorylation levels of EGFR in the AML12 hepatocytes treated with rMSA (600 μM) at different time points. F Western blot showing the protein and phosphorylation levels of EGFR, AKT, and FOXO1 in the AML12 hepatocytes treated with vehicle or rMSA (600 μM) in the absence or presence of 10 μM BDTX-189 or 10 μM Varlitinib for 6 h. G The AML-12 hepatocytes treated with or without rMSA (600 μM) for 2 h in the absence or presence of 2 μM Dasatinib were lysed; comparable amounts of total cell lysates immunoprecipitated with anti-CAV1 antibody were resolved by SDS/PAGE and visualized by the indicated antibodies. H Western blot showing the protein and phosphorylation levels of EGFR, AKT, and CAV1 in the AML12 hepatocytes transfected with siRNA against CAV1 (siCAV1-1 and siCAV1-2) for 48 h then treated with vehicle or rMSA (600 μM) in the absence or presence of 2 μM Dasatinib for 2 h. β-Actin was used as the internal reference ( A , C , D , and F – H )

Article Snippet: For the SRC signaling, after the pretreatment with vehicle, PP1 (Selleck, S7060), or Dasatinib (Selleck, S1021) for 30 min, indicated concentrations of rMSA were added at the same time in the absence or presence of SRC inhibitors.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Immunoprecipitation, SDS Page, Transfection