Journal: Inflammatory Bowel Diseases

Article Title: Overexpression of PAK1 Promotes Cell Survival in Inflammatory Bowel Diseases and Colitis-associated Cancer

doi: 10.1097/MIB.0000000000000281

Figure Lengend Snippet: Activation of AKT and mTOR signaling in IBD and CAC. A, Immunostaining of IBD and CAC samples with phospho-AKT (Thr 308). Compared with controls, epithelial p-AKT1 showed higher cytoplasmic and nuclear staining in IBD that further increased in CAC. B, IRS showed a trend toward an increase in AKT activity with the progression of disease to CAC ( P = 0.09). C, Activation of mTOR was increased in IBD samples but not in CAC as compared with controls. D, IRS scores demonstrated that mTOR activation was significantly increased in IBD (* P ≤ 0.05).

Article Snippet: Antibodies against PAK1 (#2602, cell signaling; n = 12 controls; n = 17 UC; n = 13 CD; n = 9 CAC) or p-AKT1 (Thr308; sc-135650, Santa Cruz; n = 12 controls; n = 17 UC; n = 13 CD; n = 6 CAC); p-mTOR (Ser 2448; cell signaling; n = 5 controls; n = 5 UC; n = 5 CD; n = 8 CAC) were incubated at 4°C overnight, followed by incubation with biotinylated anti-rabbit antibody and avidin–biotin–HRP complex.

Techniques: Activation Assay, Immunostaining, Staining, Activity Assay

Journal: eLife

Article Title: PH domain-mediated autoinhibition and oncogenic activation of Akt

doi: 10.7554/eLife.80148

Figure Lengend Snippet:

Article Snippet: Antibody , Akt phospho-Thr308 (Rabbit monoclonal) , Cell Signaling Technology , Cat. No.: 9275S, RRID: AB_329828 , WB (1:1000).

Techniques: Avidin-Biotin Assay, Recombinant, Software

Journal: Molecular and Cellular Biology

Article Title: Physical and Functional Interactions of the Arf Tumor Suppressor Protein with Nucleophosmin/B23

doi: 10.1128/mcb.24.3.985-996.2004

Figure Lengend Snippet: FIG. 2. Arf associates with proteins involved in ribosome biogenesis. (A) NIH 3T3 cells were infected with a retrovirus expressing TAP-tagged Arf and lysed 48 h later. Purifications were performed with lysates from cells expressing TAP-tagged Arf (lanes 2 and 3) or from uninfected cells (lane 1). TAP-tagged Arf complexes were electrophoretically resolved on denaturing gels and visualized with silver (lanes 1 and 2, 10% of purified protein loaded) or Coomassie blue (lane 3, 80% of purified protein loaded). Coomassie blue-stained bands were sequenced by mass spectrometry. (B) NIH 3T3 cells were infected with retroviruses expressing TAP-tagged full-length Arf, Arf 2-14, or Arf 2-62 and lysed 48 h later. Purifications were performed with the same amount of protein from each of the three cell lysates. Purified complexes were resolved on denaturing gels, blotted onto a polyvinylidene difluoride membrane, stained with Sypro Ruby, and visualized with a Storm imager with the blue filter. Bands corresponding to those in panel A are indicated. (C) Lysates were either left untreated (lane 1) or treated with RNase A (lane 2) prior to affinity purification from equal quantities of total protein. Recovered proteins were separated electrophoretically and stained with Coomassie blue. The lower panel shows the results of immunoblotting performed with antibodies to ribosomal protein L7.

Article Snippet: Proteins were detected with antibodies to p19Arf (33), p53 (Ab-7, Oncogene Research Products, San Diego, Calif.), p21Cip1 (C-19, Santa Cruz Biotechnology, Santa Cruz, Calif.), NPM (C-19 Santa Cruz), Flag tag (M2, Sigma Chemicals), or ribosomal protein L7 (Novus Biologicals, NB200-308).

Techniques: Infection, Expressing, Staining, Mass Spectrometry, Membrane, Western Blot

Journal: eLife

Article Title: The structural determinants of PH domain-mediated regulation of Akt revealed by segmental labeling

doi: 10.7554/eLife.59151

Figure Lengend Snippet:

Article Snippet: Antibody , Akt phospho-Thr308 (D25E6) (Rabbit monoclonal) , Cell Signaling Technology , Cat. No.: 13038S, RRID: AB_2629447 , WB (1:1000, for cell-based assays; 1:10000 for activation assays).

Techniques: Activation Assay, Avidin-Biotin Assay, Recombinant, Software