Journal: Immunity

Article Title: The Coagulation and Immune Systems Are Directly Linked through the Activation of Interleukin-1α by Thrombin

doi: 10.1016/j.immuni.2019.03.003

Figure Lengend Snippet: IL-1α Is Activated by Direct Thrombin Cleavage (A) Protein alignment showing conservation of a (K)PRS motif in diverse species. (B and C) Western blots for IL-1α showing cleavage of recombinant p33 to an ∼18kDa form by thrombin (B), and inhibition of cleavage after mutating Arg 112 to His (C). (D) N-terminal sequencing of thrombin-cleaved human p33 IL-1α detected one sequence (italic underlined) corresponding to processing between Arg 112 and Ser 113 . (E–G) IL-1-dependent IL-6 production by HeLa cells incubated with calpain cleaved (E) or thrombin cleaved (F) p33 IL-1α, ± a neutralizing IL-1α pAb (+αAb), or increasing concentrations of recombinant p17 or p18 (G). (H and I) Cleavage and activation of p33 IL-1α during clotting of platelet-rich plasma (PRP) as shown with a cleaved IL-1α-specific ELISA (H) or IL-1-dependent IL-6 production by HeLa cells (I), ± a calpain inhibitor (+Ci), or an IL-1α pAb (+αAb). (J) Mutation of Arg 112 to His prevents p33 activation during clotting of PRP. (K) Pictogram showing cleavage sites within p33 IL-1α. Data represent mean ± SEM; n = 3 (E–G, I), n = 5 (H), n = 2 (J); p = ∗ ≤0.05, ∗∗ ≤0.01, ∗∗∗ ≤0.001; NS = not significant. See also Figure S1 .

Article Snippet: Rabbit polyclonal anti-human p18 IL-1α , Innovagen , N/A.

Techniques: Western Blot, Recombinant, Inhibition, Sequencing, Incubation, Activation Assay, Coagulation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: Immunity

Article Title: The Coagulation and Immune Systems Are Directly Linked through the Activation of Interleukin-1α by Thrombin

doi: 10.1016/j.immuni.2019.03.003

Figure Lengend Snippet: Key Cell Types Contain p33 IL-1α that Can Be Cleaved by Thrombin (A and B) Flow cytometry plots and mean fluorescence intensity (MFI) for cell surface IL-1α in murine J2 macrophages treated ± LPS (A), or LPS followed by thrombin (+Tmb), ± a thrombin inhibitor (PPACK) (B). (C and D) Cleaved IL-1α-specific ELISA (C) and IL-1 activity assay (D) showing release of active p18 from the surface of LPS-treated J2 macrophages into the conditioned media by thrombin. (E and F) Human skin sections stained (brown) for IL-1α (E) or tissue factor (F). (G) Western blot for IL-1α in human keratinocyte necrotic lysates, ± a calpain inhibitor (+Ci), ± thrombin. (H) Cleaved IL-1α-specific ELISA showing thrombin cleavage of p33 IL-1α within human keratinocyte conditioned media. (I) Western blot for IL-1α showing p33 within human platelets. (J) Cleaved IL-1α-specific ELISA showing thrombin cleavage of p33 IL-1α in human platelet lysates or recombinant p33. (K) Flow cytometry for CD41 and IL-1α on resting, and collagen or collagen-related peptide (CRP) treated human platelets. Data represent mean ± SEM; n = 3 (C, D, J), n = 2 (H), n = 5 (K); p = ∗ ≤0.05, ∗∗ ≤0.01, ∗∗∗ ≤0.001. Scale bars represent 100μm. See also Figure S1.

Article Snippet: Rabbit polyclonal anti-human p18 IL-1α , Innovagen , N/A.

Techniques: Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Activity Assay, Staining, Western Blot, Recombinant

Journal: Immunity

Article Title: The Coagulation and Immune Systems Are Directly Linked through the Activation of Interleukin-1α by Thrombin

doi: 10.1016/j.immuni.2019.03.003

Figure Lengend Snippet: Generation of a Mouse Model in which IL-1α Cannot Be Activated by Thrombin (A) IL-1-dependent IL-6 production by murine fibroblasts incubated with increasing concentrations of recombinant mouse p17 or p33 IL-1α. (B) Western blot for IL-1α showing thrombin cleavage of recombinant mouse wild-type p33 to a ∼18kDa form, but less cleavage of Arg 114 His p33. (C–E) IL-1-dependent IL-6 production by murine fibroblasts incubated with increasing concentrations of recombinant mouse p17 or p18 (C), or wild-type (D) and Arg 114 His mutant (E) p33 incubated with clotting platelet-rich plasma (PRP), ± a calpain inhibitor (+Ci) and/or an IL-1α pAb (+αAb). (F) Western blot for IL-1α showing no thrombin cleavage of an Arg 114 Gln mutant p33. (G and H) IL-1-dependent IL-6 production by murine fibroblasts incubated with wild-type or mutant p33 incubated with clotting platelet-rich plasma (G) or active calpain (H). (I) Western blot for IL-1α showing equivalent expression of exogenous wild-type or mutant p33 in HeLa cells. (J) Cleaved IL-1α-specific ELISA reporting the level of calpain or thrombin processing of p33 IL-1α derived from LPS-treated control or IL-1α thrombin mutant (IL-1α TM) mBMDMs. (K) Flow cytometry for surface IL-1α on LPS-treated control or IL-1α TM mBMDMs incubated ±thrombin (+Tmb) and ± a thrombin inhibitor (PPACK). Data represent mean ± SEM; n = 3 (A, E, and J), n = 2 (C and H), n = 5 (G); p = ∗ ≤0.05, ∗∗ ≤0.01, ∗∗∗ ≤0.001; NS = not significant. See also and , and .

Article Snippet: Rabbit polyclonal anti-human p18 IL-1α , Innovagen , N/A.

Techniques: Incubation, Recombinant, Western Blot, Mutagenesis, Coagulation, Clinical Proteomics, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Control, Flow Cytometry

Journal: Immunity

Article Title: The Coagulation and Immune Systems Are Directly Linked through the Activation of Interleukin-1α by Thrombin

doi: 10.1016/j.immuni.2019.03.003

Figure Lengend Snippet: p18 IL-1α Drives Rapid Thrombopoiesis and Wound Healing, and Is Generated during Sepsis in Humans (A and B) Platelet count by flow cytometry in mice under basal conditions (A), or over time after anti-CD42b-mediated platelet depletion (B). (C) Flow cytometry for the number of CD41 + and CD61 + MKs in the bone marrow of control and IL-1α TM mice under basal conditions. (D) Cleaved IL-1α-specific ELISA reporting the level of cleaved IL-1α within the serum of control mice treated ±dabigatran (+Dabi) and IL-1α TM mice over time after anti-CD42b-mediated platelet depletion. (E–G) Representative images showing gross healing of 4 mm excisional skin wounds (E), quantitation of wound area (F) over time, and rate of closure (G) in mice as indicated. (H–K) Representative images and quantitation of Ly6G+ neutrophils (H and I) or Mac3+ macrophages (J and K) recruited to the granulation tissue underlying wounds at the times indicated. (L) ELISA data showing release of cleaved IL-1α or IL-1β from wounded skin. (M) Sandwich ELISA data showing reactivity of the p18-specific ELISA to p17, p18, or p33 IL-1α. (N and O) p18-specific ELISA data reporting level of p18 in plasma from control individuals or patients with sepsis-associated ARDS (N). Red circles indicate +VE microbiology in bronchoalveolar lavage fluid. (O) Cleaved IL-1α-specific ELISA reporting the level of cleaved IL-1α within the serum of control and IL-1α TM mice during LPS-induced endotoxemia. Data represent mean ± SEM; n = ≥4 (A, B, and D), n = 3 (C), n = ≥5 (F and G), n = ≥10 wounds (I and K), n = 20 wounds (L), n = 2 (M); p = ∗ ≤0.05, ∗∗ ≤0.01, ∗∗∗ ≤0.001; NS = not significant. See also Figure S4 and Table S2, S3.

Article Snippet: Rabbit polyclonal anti-human p18 IL-1α , Innovagen , N/A.

Techniques: Generated, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Sandwich ELISA, Clinical Proteomics

Journal: Immunity

Article Title: The Coagulation and Immune Systems Are Directly Linked through the Activation of Interleukin-1α by Thrombin

doi: 10.1016/j.immuni.2019.03.003

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-human p18 IL-1α , Innovagen , N/A.

Techniques: Plasmid Preparation, Virus, Recombinant, Red Blood Cell Lysis, Activation Assay, Staining, Lysis, Extraction, Western Blot, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Derivative Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: LMTK2-mediated Phosphorylation Regulates CFTR Endocytosis in Human Airway Epithelial Cells *

doi: 10.1074/jbc.M114.563742

Figure Lengend Snippet: Experiments demonstrating that LMTK2 localizes at the plasma membrane and co-immunoprecipitates with CFTR in human airway epithelial cells. A , schematic illustration of domain organization in CFTR and LMTK2. CFTR: TMD , transmembrane domain; NBD , nucleotide binding domain; R , regulatory domain. The R contains the phosphor-specific inhibitory Ser 737 site and other PKA consensus sites. LMTK2: TMD , transmembrane domain; KD , kinase domain. The residue Lys 168 located upstream of the Walker A motif is critical for kinase activity ( , ). MBD , myosin binding domain. Amino acid residues 567–773 mediate direct binding to myosin VI . TD , tail domain. B and C , immunoblots demonstrating similar distribution of LMTK2 at the plasma membrane of primary differentiated HBE cells and polarized human bronchial epithelial cell model CFBE41o- cells. The apical ( AP ) or basolateral ( BL ) plasma membrane ( PM ) proteins were isolated by domain-selective cell surface biotinylation in monolayers cultured on separate Transwell permeable growth supports. The whole cell lysate ( WCL ) and PM fraction of apically or basolaterally biotinylated monolayer were labeled AP and BL , respectively. Epithelial cell polarization is demonstrated by the basolateral localization of Na,K-ATPase. LMTK2 was detected at both membrane domains with AP:BL ratio of ∼1:1 when normalized for the corresponding LMTK2 abundance in WCL. WCL represents 5% of BT sample. D and E , immunoprecipitation experiments demonstrating that LMTK2 and CFTR co-immunoprecipitate in Calu-3 cells. CFTR was immunoprecipitated with the mouse monoclonal antibody M3A7 (IP CFTR, D ), and LMTK2 was immunoprecipitated with the rabbit polyclonal anti-LMTK2 antibody (IP LMTK2, E ). Mouse or rabbit non-immune IgGs were used as controls (IP IgG). WCL represents 2% of IP sample. Proteins were separated by SDS-PAGE using 7.5% gels and analyzed by immunobloting ( IB ) as indicated. All experiments were repeated three times from separate cultures with similar results.

Article Snippet: The following anti-human CFTR antibodies were used: mouse monoclonal, clone 596 (Cystic Fibrosis Foundation Therapeutics, Inc.; Chapel Hill, NC) and clone M3A7 (Millipore; Billerica, MA), and rabbit polyclonal Ab-737 (Assay Biotechnology Inc. San Francisco, CA).

Techniques: Clinical Proteomics, Membrane, Binding Assay, Residue, Activity Assay, Western Blot, Isolation, Cell Culture, Labeling, Immunoprecipitation, SDS Page