Journal: Annals of hematology

Article Title: A novel germline hyperactivating JAK2 mutation L604F.

doi: 10.1007/s00277-023-05423-y

Figure Lengend Snippet: Fig. 1 The pedigree of the fam- ily with the JAK2 L604F muta- tion. The patient and her sister have the L604F mutation (blue) in a homozygous state, and her parents have heterozygous L604F. The germline L604F probably arises from a common ancestor pair (encircled in blue). The presence of the mutation in the nontested family members is presumed (light blue) as a highly likely way to yield the results obtained in the tested members

Article Snippet: Plasmids with JAK2 wild-type (WT) or JAK2 V617F were constructed by PCR-based techniques of molecular cloning by incorporating JAK2 WT or JAK2-V617F sequences from pDONR223 plasmids containing respective genes [Addgene plasmids # 23,915 and # 81,756 [23, 24]] into plasmid pEGFP-N2 (originally Clontech, Mountain View, CA, USA) designed for exogenous expression of proteins with a green fluorescent protein (eGFP) tag.

Techniques: Mutagenesis

Journal: Annals of hematology

Article Title: A novel germline hyperactivating JAK2 mutation L604F.

doi: 10.1007/s00277-023-05423-y

Figure Lengend Snippet: Fig. 3 Effect of endogenous JAK2 mutations in HeLa cells. Western blot analysis of HeLa cells with V617F or L604F JAK2 mutations introduced by CRISPR. The expected gene modification was confirmed by sequencing. Eight independent harvests were performed for each modified subline and the wild type (WT) parental line. The cell lysates were analyzed in groups – each western-blot membrane contained 4 WT samples and 4 samples from one mutated subline. The measured band intensities were normal- ized to ACTIN and related to the mean value from WT samples included in the given membrane (100%). Top: repre- sentative examples of JAK2 and pJAK2 Tyr1007/1008 signals. ACTIN was used as the loading control. Bottom: means ± SD of the relative band intensities from 8 independent samples for each JAK2 variant. The differences between modified sublines and the WT control were evaluated by unpaired Student´s t test (** p < 0.01, *** p < 0.001)

Article Snippet: Plasmids with JAK2 wild-type (WT) or JAK2 V617F were constructed by PCR-based techniques of molecular cloning by incorporating JAK2 WT or JAK2-V617F sequences from pDONR223 plasmids containing respective genes [Addgene plasmids # 23,915 and # 81,756 [23, 24]] into plasmid pEGFP-N2 (originally Clontech, Mountain View, CA, USA) designed for exogenous expression of proteins with a green fluorescent protein (eGFP) tag.

Techniques: Western Blot, CRISPR, Modification, Sequencing, Membrane, Control, Variant Assay

Journal: Chemistry & biology

Article Title: Selecting agonists from single cells infected with combinatorial antibody libraries.

doi: 10.1016/j.chembiol.2013.04.012

Figure Lengend Snippet: Figure 5. Signal Transduction by Agonist Antibodies JAK2 was purified from cell lysates using affinity to anti-JAK2 agarose and its phosphorylation was detected with western blotting using anti- phosphotyrosine antibodies. Phosphorylation of STAT3, STAT5, Akt, and MAPK induced by agonist antibodies or rhTPO stimulation was detected with direct western blotting of cell lysates. See also Figure S6.

Article Snippet: The immunoprecipitates were analyzed with western blotting using anti-phospho-JAK2 (Tyr1007/1008) (C80C3) antibody (Cell Signaling Technology, catalog no. 3776) and anti-total JAK2 antibody (C-14) (Santa Cruz Biotechnology, catalog no. sc-34479).

Techniques: Transduction, Phospho-proteomics, Western Blot

Journal: Discover oncology

Article Title: Hepatoma cell-derived exosomal SNORD52 mediates M2 macrophage polarization by activating the JAK2/STAT6 pathway.

doi: 10.1007/s12672-024-01700-y

Figure Lengend Snippet: Fig. 3 SNORD52 mediates M2 macrophage polariza- tion and activates JAK2/ STAT6 pathway. A Following transfection of OE-SNORD52/ OE-NC in THP-1 and U937 for 48 h, the mRNA expression of SNORD52 was detected by qRT-PCR. ****P < 0.0001 vs. OE-NC. B Following trans- fection of OE-SNORD52/ OE-NC in THP-1 and U937 for 48 h, the protein levels of Arginase-1 and CD163 were determined through Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001 vs. OE-NC. C The ratio of CD206-positive or CD163-positive cells following transfection of OE-SNORD52/ OE-NC was analyzed by flow cytometry. ***P < 0.001 vs. OE-NC. D Following transfec- tion of OE-SNORD52/OE-NC in THP-1 and U937 for 48 h, the levels of JAK2/STAT6 pathway-related proteins were determined through Western blotting. ***P < 0.001 vs. OE-NC. E The IL-4 and IL-13 treated THP-1/U937 mac- rophages were co-incubated with SNORD52 ASO-1/-2 or ASO-NC for 48 h, and then the mRNA expression of SNORD52 was detected by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001 vs. ASO-NC. F The IL-4 and IL-13 treated THP-1/U937 mac- rophages were co-incubated with SNORD52 ASO-1/-2 or ASO-NC for 48 h, and then the protein levels of Arginase-1 and CD163 were determined through Western blotting. ***P < 0.001 vs. ASO-NC. G The ratio of CD206-positive or CD163-positive cells following transfection of SNORD52- ASO-1/-2 or ASO-NC was analyzed by flow cytometry. ***P < 0.001 vs. ASO-NC. (H) The IL-4 and IL-13 treated THP-1/ U937 macrophages were co-incubated with SNORD52 ASO-1/-2 or ASO-NC for 48 h, and then the levels of JAK2/STAT6 pathway-related proteins were determined through Western blotting. ***P < 0.001 vs. ASO-NC. ns: no significance

Article Snippet: Meanwhile, the primary antibodies Arginase-1, CD163, and STAT6 used in Western blotting were also purchased from Proteintech, while other primary antibodies including JAK2, p-JAK2, p-STAT6, exosome-positive markers (CD81 and TSG101), and the corresponding HRP-conjugated secondary antibodies were available from Abcam (Cambridge, UK).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Incubation

Journal: Discover oncology

Article Title: Hepatoma cell-derived exosomal SNORD52 mediates M2 macrophage polarization by activating the JAK2/STAT6 pathway.

doi: 10.1007/s12672-024-01700-y

Figure Lengend Snippet: Fig. 5 Huh7 cells-derived exosomal SNORD52 mediates M2 macrophage polarization through activating JAK2/STAT6 pathway. A Following the establishment of co-culture model, AG-490 (a specific JAK2 inhibitor) was added and the levels of JAK2/STAT6 pathway-related pro- teins in receptor THP-1 macrophages were measured by Western blotting. B Following the establishment of co-culture model, AG-490 (a specific JAK2 inhibitor) was added and the ratio of CD206-positive or CD163-positive THP-1 macrophages was analyzed by flow cytometry. **P < 0.01, ***P < 0.001. ns: no significance

Article Snippet: Meanwhile, the primary antibodies Arginase-1, CD163, and STAT6 used in Western blotting were also purchased from Proteintech, while other primary antibodies including JAK2, p-JAK2, p-STAT6, exosome-positive markers (CD81 and TSG101), and the corresponding HRP-conjugated secondary antibodies were available from Abcam (Cambridge, UK).

Techniques: Derivative Assay, Co-Culture Assay, Western Blot, Flow Cytometry