Journal: The Journal of Physiology

Article Title: Bile acids induce necrosis in pancreatic stellate cells dependent on calcium entry and sodium‐driven bile uptake

doi: 10.1113/JP272774

Figure Lengend Snippet: A , mRNA expression of selected genes encoding bile acid transporters in human pancreatic stellate cells (hPSCs) and human hepatocytes. PCR products for the sodium–taurocholate cotransporting polypeptide (NTCP, slc10A1) and sodium independent transporters, OATP4A1 ( slco4A1 ) and OATP1B3 (slco1B3) , are present in human hepatocytes; hPSCs express slc10A1 and slco4A1 , but not slco1B3 . B , immunoblotting shows expression of NTCP in different tissues; lines from the left: mass marker, hPSC, four positive controls (human liver and mouse pancreas, liver and kidney), two negative controls (mouse spleen and lung). C , immunohistofluorescence (IHF) staining for NTCP in fixed mouse tissue sections: the pancreas (upper panel) and the liver (lower panel). From the left: DAPI (blue), NTCP (white), overlay images (white arrows indicate PSCs), and transmitted light images. Insets show corresponding staining in controls without primary antibody. D , double IHF staining for NTCP and the bradykinin receptor B2 (BDKRB2). From the upper left: DAPI (blue), NTCP (green), BDKRB2 (red), fluorescence overlay (PSCs are indicated by white arrows). E and F , the comparison of homological sequences of three Na + ‐dependent mouse transporters (NTCP, ASBT and SOAT) with immunogens used for generation of the antibodies for immunoblotting ( E ) and IHF ( F ). Red letters represent amino acids identical to those in the immunogen sequence; blue letters are similar amino acids, and black letters show amino acids that are different. Only mouse NTCP sequences share high sequence identity with the immunogens.

Article Snippet: Human liver tissue lysate was obtained from Novus Biologicals, Littleton, CO, USA.

Techniques: Expressing, Western Blot, Marker, Immunohistofluorescence, Staining, Fluorescence, Comparison, Sequencing

Journal: Thrombosis research

Article Title: Thrombomodulin enhances complement regulation through strong affinity interactions with factor H and C3b-Factor H complex.

doi: 10.1016/j.thromres.2016.07.017

Figure Lengend Snippet: Fig. 1. Binding affinity of FH to recombinant human TM analogs. (A) A ligand binding assay confirmed the binding of FH (1,2 and 5 μg/ml) to immobilized TM1. Data points represent mean ± SD of triplicate determinations (error bars depicted for each point) within one binding assay. FH was flowed over polyclonal antibody-captured recombinant human thrombomodulin (B) TM1 and (C) TM2 at concentrations between 1.55 nM and 1.6 μM and equilibrium binding response was measured. Affinity (KD) was determined by steady-state analysis in duplicate for TM1 KD 276 ± 60 nM and TM2 KD 114 ± 25 nM (mean ± SD, n = 2).

Article Snippet: Polyclonal antiTM antibody and the two human recombinant soluble TM were from R & D Systems Europe Ltd. (Abingdon, UK) and Abcam plc (Cambridge, UK); thefirstwas produced in amousemyeloma cell line (NS0-derived) with sequence: Ala19-Ser515, with C-terminal 6-His-tag, TM1.

Techniques: Binding Assay, Recombinant, Ligand Binding Assay

Journal: Thrombosis research

Article Title: Thrombomodulin enhances complement regulation through strong affinity interactions with factor H and C3b-Factor H complex.

doi: 10.1016/j.thromres.2016.07.017

Figure Lengend Snippet: Fig. 2. Binding affinity of C3b and C3b-FH to recombinant human TM analogs. C3b was flowed over (A) TM1 and (B) TM2 at concentrations between 4.4 nM and 5 μM and affinity was determined by 1:1 binding model for TM1 KD 1.13 ± 0.09 μM and TM2 KD 1.16 ± 0.02 μM (mean ± SD, n = 2). Preformed equimolar C3b-FH complex was flowed over (C) TM1 and (D) TM2 at concentrations between 7.8 nM and 800 nM and affinity was determined by 1:1 binding model for TM1 KD ~91 nM and TM2 KD ~76 nM (n = 1).

Article Snippet: Polyclonal antiTM antibody and the two human recombinant soluble TM were from R & D Systems Europe Ltd. (Abingdon, UK) and Abcam plc (Cambridge, UK); thefirstwas produced in amousemyeloma cell line (NS0-derived) with sequence: Ala19-Ser515, with C-terminal 6-His-tag, TM1.

Techniques: Binding Assay, Recombinant

Journal: Thrombosis research

Article Title: Thrombomodulin enhances complement regulation through strong affinity interactions with factor H and C3b-Factor H complex.

doi: 10.1016/j.thromres.2016.07.017

Figure Lengend Snippet: Fig. 4. TM regulates complement activity in serum. Activated sheep erythrocytes (ShEA) were incubated with normal human serum (NHS) spiked with 100 nM human recombinant TM (circles) compared to NHS alone (squares) and lysis was developed. Data points represent mean ± SD of three determinations (error bars depicted for each point). Lysis was calculated by measuring hemoglobin release as a percentage of the controls. Nonlinear regression curves (A, C) and CH50 log-log plot (B, D) are shown for TM1 (A,B) and TM2 (C,D), respectively. (E) Summary of CH50 values (NHS volume in μl at 50% hemolysis) each for TM1 (n = 2) and TM2 (n = 2) in NHS compared to NHS only and corresponding p values analyzed in independent assays. P values were calculated using a two-tailed unpaired t-test.

Article Snippet: Polyclonal antiTM antibody and the two human recombinant soluble TM were from R & D Systems Europe Ltd. (Abingdon, UK) and Abcam plc (Cambridge, UK); thefirstwas produced in amousemyeloma cell line (NS0-derived) with sequence: Ala19-Ser515, with C-terminal 6-His-tag, TM1.

Techniques: Activity Assay, Incubation, Recombinant, Lysis, Two Tailed Test

Journal: Thrombosis research

Article Title: Thrombomodulin enhances complement regulation through strong affinity interactions with factor H and C3b-Factor H complex.

doi: 10.1016/j.thromres.2016.07.017

Figure Lengend Snippet: Fig. 5. Complement regulation by human recombinant TM456 fragment. (A) TM456 binding assay confirmed the binding of FH (1,2 and 5 μg/ml) to immobilized TM456. Data points represent mean ± SD of triplicate determinations (error bars depicted for each point) within one binding assay. (B) Serial dilution of TM456 (0.125-2 μM) added to 1% ΔΒH-NHS with constant FB and FH and ShEA to develop lysis in CFD. In the absence of TM456, 85% lysis was achieved by 1% ΔΒH-NHS, whereas the maximum dose of 2 μM reduced lysis to 61%. (C) Serial dilutions of FH (0.001-1 μM) was added to TM456-sufficient (2 μM) or deficient 1% ΔBH-NHS and lysis developed in ShEA, with FB in CFD. FH-H50 was 15 ± 4 nM in the presence of TM456 (circles) and 41 ± 3 nM (squares) in its absence (p = 0.018). (D) The maximum response (% Lysis) was reduced for TM456-suffient 1% ΔBH-NHS (circles, Max%Lysis 60.9 ± 2.8) compared to 1% ΔBH-NHS alone (squares, Max%Lysis, 73.6 ± 0.5, p = 0.018). Data points represent mean ± SD of three determinations (error bars depicted for each point, the assay was repeated twice with identical results).

Article Snippet: Polyclonal antiTM antibody and the two human recombinant soluble TM were from R & D Systems Europe Ltd. (Abingdon, UK) and Abcam plc (Cambridge, UK); thefirstwas produced in amousemyeloma cell line (NS0-derived) with sequence: Ala19-Ser515, with C-terminal 6-His-tag, TM1.

Techniques: Recombinant, Binding Assay, Serial Dilution, Lysis

Journal: Oncotarget

Article Title: KIAA1114, a full-length protein encoded by the trophinin gene, is a novel surface marker for isolating tumor-initiating cells of multiple hepatocellular carcinoma subtypes

doi:

Figure Lengend Snippet: ( A and B ) Flow cytometric analysis of surface KIAA1114 expression in human CC cell lines (A) and AFP + and AFP − HCC cell lines (B) . (C) Immunoblot analysis of tissue lysates from normal liver (N), patient tumor (PT), and its adjacent normal tissue from the same donor (PN). β-actin served as an internal control.

Article Snippet: Human liver whole tissue lysate and paired primary tumor and normal tissues were purchased from Novus Biologicals.

Techniques: Expressing, Western Blot, Control