Journal: PLoS ONE

Article Title: Interferon-γ Suppresses Intestinal Epithelial Aquaporin-1 Expression via Janus Kinase and STAT3 Activation

doi: 10.1371/journal.pone.0118713

Figure Lengend Snippet:

Article Snippet: Phosphorylated AKT (Cat# AF887, R & D Systems, Minneapolis, MN) , 5% BSA in TTBS , 1:400 , goat anti-rabbit.

Techniques: Blocking Assay, Concentration Assay

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) Flag-tagged AKT was transiently overexpressed in HEK293T cells semi-stably expressing either V5-DHR1L or V5-DHR2. Cells were then either treated with vehicle control (DMSO, 1:1000) or with MK2206 (10 μM) for 16 h before being collected and lysed. Immunoprecipitation was performed using anti-V5 beads and complexes were resolved on an SDS-PAGE gel for Western analysis. ( B ) HEK293T cells semi-stably expressing either V5-DHR1L or V5-DHR2 were grown in full-serum or serum starved for 16 h, collected, lysed then used for immunoprecipitation and western blot analysis as described in ( A ). Proximity Ligation Assays (PLA) were performed to determine the number of direct interactions between endogenous Dock7 and either ( C ) endogenous AKT or ( D ) phospho-AKT in HeLa cells, respectively. ( E ) Cirspr-Cas9 Dock7 KO and WT HeLa cells were seeded and allowed to recover for 24 h. Media was then changed to either full serum media or serum-free media for 24 h. Serum-starved cells were then treated with either vehicle control DMSO (10 µl), Okadaic Acid (10 nM), or Calyculin A (50 nM) for 1 h before being collected for Western blot analysis. ( F ) Proximity ligation assay was performed on Cirspr-Cas9 Dock7 KO and WT HeLa cells to measure the number of complexes formed between AKT and PHLPP in the presence and absence of Dock7.

Article Snippet: Primary antibodies for V5-tag (Invitrogen, MA5-15243) and pan AKT (R&D Systems, MAB2055) conjugated to Duolink PLA Multicolor Probemaker Kit Green (Sigma Aldrich, DUO96020), while pan AKT (R&D Systems, MAB2055) and p-AKT S473 (R&D Systems, AF887) were conjugated with the Probemaker Kit Red (Sigma-Aldrich, DUO96010).

Techniques: Stable Transfection, Expressing, Control, Immunoprecipitation, SDS Page, Western Blot, Ligation, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A and B ) Dock7 was semi-stably knocked down using shRNA and either an empty vector or vectors containing the limit DHR domains were expressed using lentiviral system. Cells were then seeded in either ( A ) soft agar suspension and counted two weeks later or ( B ) in 100 mm plates for protein analysis using western blotting. ( C ) Lentiviral transduction system was used to overexpress the specified domains and their mutants in HeLa WT cells. Cells expressing each plasmid were selected with antibiotics for 3-5 days, seeded, and allowed to recover for 24 h. Media was changed to serum-free media and cells were collected after 24 h for Western blot analysis. ( D ) Semi-stable HeLa cells overexpressing either an empty vector, DHR1L, or its C2 mutant were seeded, allowed to recover, and then allowed to grow for four days in serum-free conditions. Cells were then trypsin-treated and counted. ( E - G ) Dock7 was knocked down using shRNA in MDA-MB-231 cells, and then the specified constructs were overexpressed. Cells were then either grown in complete media or starved for 20-24 h. Proximity ligation assays (PLA) were used determine the number of interactions between endogenous AKT or phospho-AKT and each Dock7 construct. Western blots are representative of three separate biological replicates. Survival assays were performed in triplicates. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in (DG) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01, ∗p < 0.1, and not significant (n.s.)

Article Snippet: Primary antibodies for V5-tag (Invitrogen, MA5-15243) and pan AKT (R&D Systems, MAB2055) conjugated to Duolink PLA Multicolor Probemaker Kit Green (Sigma Aldrich, DUO96020), while pan AKT (R&D Systems, MAB2055) and p-AKT S473 (R&D Systems, AF887) were conjugated with the Probemaker Kit Red (Sigma-Aldrich, DUO96010).

Techniques: Stable Transfection, shRNA, Plasmid Preparation, Suspension, Western Blot, Transduction, Expressing, Mutagenesis, Construct, Ligation