Journal: Nature cardiovascular research

Article Title: Angiopoietin-TIE2 feedforward circuit promotes PIK3CA-driven venous malformations.

doi: 10.1038/s44161-025-00655-9

Figure Lengend Snippet: Fig. 6 | Increased TIE2 phosphorylation and SMC coverage in Pik3ca-driven VM in mice. a, PLA staining of activated TIE2 on ear skin paraffin sections from Pik3caH1047R;Vegfr1-CreERT2 and Cre− littermate Ctrl mice, detected using pTyr and TIE2 antibodies. DAPI marks cell nuclei. b, Quantification of PLA signals within PECAM1+ blood vessels. Data represent the number of PLA dots per μm2 of PECAM1+ vessel area, mean ± s.d. (n = 8 (Ctrl) and n = 26 (Pik3caH1047R) vessels from four mice per genotype, unpaired two-tailed Student’s t-test, ****P = 0.0000054). c, Immunofluorescence of ear skin paraffin sections from Pik3caH1047R;Vegfr1- CreERT2 and Cre− littermate Ctrl mice using phospho-TIE2 antibodies. d, Phospho- TIE2 signal within EMCN+ vessels, represented as corrected total cell fluorescence (CTFC) of EMCN+ vessel area. Data points represent pTIE2 CTFC, mean ± s.d. (n = 20 (Ctrl) and n = 31 (Pik3caH1047R) vessels from two mice per genotype, unpaired two-tailed Student’s t-test, ****P = 0.000096; Extended Data Fig. 8d). e, Whole-mount immunofluorescence of ear skin from Pik3caH1047R;

Article Snippet: Human paraffin sections (5 μm) were treated as described above and antibodies used were rabbit pTyr (P-Tyr-1000 MultiMab, Cell Signaling, 8954, dilution 1:200) and goat anti-human TIE2 (R&D Systems, AF313, dilution 1:200).

Techniques: Phospho-proteomics, Staining, Two Tailed Test, Immunofluorescence, Fluorescence

Journal: Nature cardiovascular research

Article Title: Angiopoietin-TIE2 feedforward circuit promotes PIK3CA-driven venous malformations.

doi: 10.1038/s44161-025-00655-9

Figure Lengend Snippet: Fig. 7 | Increased TIE2 phosphorylation in human PIK3CA-driven VMs. a, H&E-stained paraffin sections of cutaneous VMs from individuals with PIK3CA (top) or TEK (below) mutations. Areas defined as non-lesional (NL) and VM lesions are indicated. b, PLA staining of activated TIE2, detected using pTyr and TIE2 antibodies, in representative vessels from indicated NL and VM regions. DAPI marks cell nuclei. c, Quantification of PLA signals within PECAM1+ veins, represented as mean PLA dots per EC nucleus ± s.d. (n = 7 (PIK3CA), n = 6 (TEK), n = 2 (Ctrl) and n = 5 (NL) individuals, with symbols indicating different mutations; ordinary one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test, **P(PIK3CA VM versus Ctrl vein) = 0.0027 and **P(TEK VM versus Ctrl vein) = 0.0078; Extended Data Fig. 7). d, Representative immunofluorescence image (left) and quantification (right) showing the

Article Snippet: Human paraffin sections (5 μm) were treated as described above and antibodies used were rabbit pTyr (P-Tyr-1000 MultiMab, Cell Signaling, 8954, dilution 1:200) and goat anti-human TIE2 (R&D Systems, AF313, dilution 1:200).

Techniques: Phospho-proteomics, Staining, Comparison, Immunofluorescence

Journal: Cell Reports

Article Title: A single amino acid in the Salmonella effector SarA/SteE triggers supraphysiological activation of STAT3 for anti-inflammatory gene expression

doi: 10.1016/j.celrep.2025.115530

Figure Lengend Snippet:

Article Snippet: Anti-pTYR-1000 monoclonal pool , CST , Cat#8954; RRID: AB_2687925.

Techniques: Virus, Recombinant, Phospho-proteomics, Mutagenesis, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Genome Wide, Mass Spectrometry, Cell Culture, Software

Journal: The EMBO Journal

Article Title: PKM2 functions as a histidine kinase to phosphorylate PGAM1 and increase glycolysis shunts in cancer

doi: 10.1038/s44318-024-00110-8

Figure Lengend Snippet: ( A ) Inhibition of PGAM1 tyrosine phosphorylation reduces PKM2 interaction. Left: A549 cells were transfected with Flag-PGAM1 for 48 h. Flag (PGAM1)-associated proteins were immunoprecipitated and immobilized on the agarose beads coated with flag antibody, and then respectively treated with or without protein tyrosine phosphatase 1B (PTP1B, 1 μg/μL) for 30 min. The protein complex was eluted and analyzed by WB. Right: Quantification of IP. Relative levels of pTyr, PKM2 or 3-pHis were normalized to that of Flag (PGAM1) for each group. ( B ) PGAM1 is phosphorylated at Y119 analyzed by MS. PGAM1 protein immunoprecipitated from A549 cells was trypsinized and then analyzed by MS. MS data were processed with the Byonic engine, and the peptide containing phosphorylated Y119 was identified. The y and b fragmentations were used to map the phosphorylation site to the Tyr indicated in red. ( C , D ) PGAM1 Y119 phosphorylation determines PKM2 association. Left: A549 cells were transfected with Flag-PGAM1 and its mutants for 48 h. Flag (PGAM1)-associated proteins were immunoprecipitated and analyzed by WB. Right: Quantification of IP. Relative levels of pTyr (PGAM1) or PKM2 were normalized to that of Flag (PGAM1) for each group. ( E , F ) PGAM1 Y119F mutation blocks PKM2-mediated PGAM1 H11 phosphorylation. In vitro kinase assay was carried out with recombinant GST-PKM2, His-PGAM1 and its Y119F mutant in the presence of PEP. ( E , top) PGAM1 H11 phosphorylation level was detected by WB. ( E , bottom) Quantification of WB. Relative levels of 3-pHis were normalized to that of His (PGAM1) for each group. ( F ) PGAM1 activity was analyzed by commercially available kits. ( G ) Tyrosine 119 is conserved through mammals. Amino acid sequences that cross Y119 of PGAM1 and its homolougues from various species. Y119 or the corresponding site is highlighted in red. ( H ) PGAM1 Y119 phosphorylation is more evident in tumor cells. Left: From HBE-2, A549 and H1299 cells, PGAM1-associated proteins were immunoprecipitated and analyzed by WB. Right: Quantification of IP. Relative levels of pY119 were normalized to that of PGAM1 for each group. ( I ) PGAM1 WT and Y119F mutant no longer have impacts on PGAM1 H11 phosphorylation in the absence of PKM2. Left: Flag-PGAM1 WT or Y119F mutant was respectively transfected into normal A549 or PKM2-depleted A549 cells for 48 h. Flag (PGAM1)-associated proteins were immunoprecipitated and analyzed by WB. Right: Quantification of IP. Relative levels of pY119, PKM2 or 3-pHis were normalized to that of Flag (PGAM1) for each group. Data information: for WB in ( A , C – E , H , I ), one representative experiment out of three is shown. For IP in ( C , D , H , I ), IgG served as a negative control. Data were represented as mean ± SD ( n = 3) with significance determined by one-way ( C – E , F , H , I ) or two-way ( A ) ANOVA test; **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns nonsignificant. .

Article Snippet: The primary antibodies used in this study include anti-PGAM1 (1:200 for WB and 1:10 for IP, Santa Cruz, 130334), anti-PKM2 (1:1000 for WB and 1:50 for IP, CST, 4053 S), anti-PKM1 (1:1000 for WB, CST, 7067), anti-NME1 (1:1000 for WB, Abclonal, A8802), anti-NME2 (1:1000 for WB, Abclonal, A7443), anti-phospho-tyrosine (pTyr) (1:1000 for WB, CST, 8954), anti-Flag (1:1000 for WB and 1:50 for IP, Sigma, F1804), anti-Actin (1:1000 for WB, Sigma, A4700), anti-ENO1 (1:1000 for WB and 1:20 for IP, CST, 3810), anti-PCK2 (1:1000 for WB, SAB, College Park, MD, USA, A11526), anti-His (1:1000 for WB, TransGen, 32301) and anti-GST (1:1000 for WB, TransGen, ab2739), anti-HA (1:1000 for WB, TransGen, HT301-01), anti-3-pHis (1:1000 for WB, Millipore, MABS1351), anti-Tubulin (1:1000 for WB, TransGen, HC101-01), anti-EGFR (1:1000 for WB, Abclonal, A23452), anti-pEGFR (1:1000 for WB, SAB, 11220), anti-STAT3 (1:1000 for WB, CST, 9139S), anti-pSTAT3 (1:1000 for WB, CST, 9145S), anti-c-Kit (1:1000 for WB, Abclonal, A0357), anti-pc-Kit (1:1000 for WB, Abclonal, AP0586), anti-Src (1:1000 for WB, Abclonal, A11707), anti-pSrc (1:1000 for WB, Abclonal, AP1377), anti-FGFR1 (1:1000 for WB, Abclonal, A21219), anti-pFGFR1 (1:1000 for WB, Abclonal, AP1317), anti-PDGFRA (1:1000 for WB, Abclonal, A2103), anti-pPDGFRA (1:1000 for WB, Abclonal, AP0615), anti-Ki-67 (1:100 for IHC, CST, 34330).

Techniques: Inhibition, Phospho-proteomics, Transfection, Immunoprecipitation, Tandem Mass Spectroscopy, Mutagenesis, In Vitro, Kinase Assay, Recombinant, Activity Assay, Negative Control