aavlea1 expression plasmid pet15b-aavlea1  (Addgene inc)

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: pLenti-V5-p53 (plasmid 22945), pET15b-p53 (plasmid 24859), lentiCRISPR v2 (plasmid 52961), MAC-STA5A (plasmid 167799) were purchased from Addgene. pGL-4.53 (plasmid E6681) was purchased from Promega. pAX2, pMD2G, Pcl-Eco, HA-Ub, MSCV-IRES-GFP-Stat5 WT; dominant-negative, and constitutively active mutant plasmids, MSCV-IRES-GFP-Jak2 WT, and V617F mutant plasmids were previously reported ( , ). pCMV-Flag-PPIL2, pNL1.1- PPIL2 and mutant promoter plasmids, prokaryotic expression vectors pET15b-His-p53 (1-97), 98-292, 293-393, pGEX6p-3-GST-PPIL2 (1-520), 1-190, and 275-435 were individually cloned.

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

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 Ppil2 in bone marrow Ter119 + , CD11b + , and Gr1 + cells from WT and Jak2 V617F -knockin mice. Hsc70 was used as a loading control. ( B ) Schematic illustration of experimental design where c-Kit + bone marrow cells from WT or Jak2 V617F mice were transduced with control or CRISPR-Ppil2 sgRNA, followed by transplantation into lethally irradiated recipient mice. The recipient mice were examined for hematologic phenotypes 8 weeks after transplant. ( C ) Complete blood count of mice from B . WT+Control: n = 10, Jak2 V617F +Control: n = 7, Jak2 V617F +sgPpil2: n = 7. ( D ) Quantification of spleen weight of mice from B . ( E ) Western blotting of Ppil2 and p53 expression in the bone marrow of indicated mice in B . ( F ) Representative flow cytometric analyses of terminal erythropoiesis using CD44 and forward scatter as markers. Populations I-VI represent proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts, reticulocytes, and mature RBCs, respectively. ( G and H ) Quantification of different stages of erythroblasts by flow cytometric analyses as F from bone marrow ( G ) and spleen ( H ). ( I ) Quantification of HSPCs by flow cytometric analyses from the bone marrow in mice from B . HPC: lineage negative, c-Kit + hematopoietic progenitor cells. GMP, granulocyte-macrophage progenitor; CMP, common myeloid progenitor; MEP, megakaryocyte-erythrocyte progenitor; LSK, Lin – Sca1 + cKit + cells; LT-HSC, long-term HSC; ST-HSC, short-term HSC; MPP, multipotential progenitor. ( J ) Representative H&E staining of the indicated organs of the indicated mice from B . Scale bars: 100 μm. The comparison among multiple groups was evaluated with 1-way ANOVA ( C , D , I , G , and H ). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: pLenti-V5-p53 (plasmid 22945), pET15b-p53 (plasmid 24859), lentiCRISPR v2 (plasmid 52961), MAC-STA5A (plasmid 167799) were purchased from Addgene. pGL-4.53 (plasmid E6681) was purchased from Promega. pAX2, pMD2G, Pcl-Eco, HA-Ub, MSCV-IRES-GFP-Stat5 WT; dominant-negative, and constitutively active mutant plasmids, MSCV-IRES-GFP-Jak2 WT, and V617F mutant plasmids were previously reported ( , ). pCMV-Flag-PPIL2, pNL1.1- PPIL2 and mutant promoter plasmids, prokaryotic expression vectors pET15b-His-p53 (1-97), 98-292, 293-393, pGEX6p-3-GST-PPIL2 (1-520), 1-190, and 275-435 were individually cloned.

Techniques: Western Blot, Knock-In, Control, Transduction, CRISPR, Transplantation Assay, Irradiation, Expressing, Staining, Comparison