Journal: The Journal of Clinical Investigation

Article Title: PPIL2 is a target of the JAK2/STAT5 pathway and promotes myeloproliferation via degradation of p53

doi: 10.1172/JCI181394

Figure Lengend Snippet: ( A ) Western blotting of indicated proteins following IP of anti-Flag with cell lysate from HEK293T cells transfected with control or Flag-PPIL2. HSC70 was used as a loading control. ( B ) IP of anti-V5 with cell lysate from day 7 cultured human HUDEP-2 cells transduced with V5-p53 followed by Western blotting of indicated proteins. ( C ) Western blotting of indicated proteins following endogenous IP of anti-Ppil2 with cell lysate from mouse bone marrow Ter119 + cells. Hsc70 was used as a loading control. ( D ) GST pulldown assays using recombinant GST-PPIL2 with His-p53. ( E ) His-p53 pulldown of GST-tagged PPIL2. ( F ) Western blotting of indicated proteins in HEK293T cells transfected with indicated constructs treated with or without MG132 (1 mM) for 6 hours. ( G ) Western blotting of indicated proteins in HEK293T cells transfected with vector or Flag-PPIL2 treated with or without 2 μg/mL CHX for the indicated time. ( H ) Densitometric analysis of relative p53 protein levels in G using ImageJ software. ( I ) Western blotting of indicated proteins in HEK293T cells transiently transduced with control or CRISPR-PPIL2 sgRNA in the presence of 2 μg/mL CHX over indicated time. ( J ) Western blotting of indicated proteins after anti-p53 IP of HEK293T cells transfected with control or Flag-PPIL2. ( K–M ) Western blotting following anti-V5 pulldown from lysates of HEK293T cells transduced with the indicated constructs. ( N ) Western blotting analysis showing expression levels of Ppil2 and p53 in WT or p53 knockout mice bone marrow lineage-negative cells infected with control or CRISPR-Ppil2 sgRNA and cultured in EPO medium. Hsc70 was used as a loading control. ( O ) Proliferation assays of cells from N cultured for 48 hours. ( P ) Schematic illustration of experimental design. ( Q ) Western blotting analyses of Ppil2 and p53 expression in the bone marrow of 3-week-old mice transplanted with donor cells transduced with the indicated constructs. Hsc70 was used as a loading control. The comparison among multiple groups was evaluated with 1-way ANOVA ( O ). ** P < 0.01.

Article Snippet: Briefly, for erythroblast differentiation, purified lineage-negative cells were cultured in IMDM containing 15% FBS (StemCell Technologies), 1% detoxified BSA (StemCell Technologies), 200 μg/mL holo-transferrin (MilliporeSigma), 10 μg/mL recombinant human insulin (MilliporeSigma), 2 mM L-glutamine, 10 –4 M β-mercaptoethanol, and 2 U/mL recombinant human EPO (Amgen).

Techniques: Western Blot, Transfection, Control, Cell Culture, Transduction, Recombinant, Construct, Plasmid Preparation, Software, CRISPR, Expressing, Knock-Out, Infection, Comparison