anti hgf polyclonal neutralizing antibody  (R&D Systems)

Journal: Aging Cell

Article Title: Age‐related nitration/dysfunction of myogenic stem cell activator HGF

doi: 10.1111/acel.14041

Figure Lengend Snippet: High‐frequency (HF) stretch does not activate satellite cells. (a) The experimental scheme for stretch cultures. Primary cultures of rat satellite cells received one of three patterns of 25% cyclic stretch for 1 h beginning at 24‐h post‐plating followed by pulse labeling with BrdU for 2 h just prior to the 48‐h, time‐point of the BrdU‐incorporation assay for activation. Conditioned media from 1‐h stretch cultures were also evaluated by western blotting and BrdU‐incorporation. (b) Three cyclic‐stretch patterns, LF, LFH, and HF, differed in intensity, as determined by stretch frequency (5.0 or 8.6 cycles/min) with/without 5‐s stretch‐retention, at 5‐s intervals. (c) Cell activation response to three cyclic‐stretch patterns. Satellite cell cultures were assayed for cell activation (BrdU‐positive cell percentage) at 48‐h post‐plating. CNT, negative control unstretched culture in DMEM‐10% HS ( open bars a and f ); positive controls, cultures with 2.5 ng/mL recombinant HGF for 24 h beginning 24‐h post‐plating ( gray bars b and g ) and for 23 h beginning 25‐h ( open bar h ) before fixation. Stretch cultures: 1‐h LFH, LF, and HF stretch ( black bars c and d , and red bar e , respectively, in the left column). The experiments were repeated using 3–6 rats independently to confirm the reproducibility of the results (panel c inset). Red bar i , HF‐stretch culture again (under the same experimental condition as bar e ); red bars j and k , 1‐h HF stretch followed by administration of 2.5 ng/mL and 10 ng/mL HGF for 23 h after stretch, respectively. (d) Immunoblotting analysis of HGF in conditioned media from 1‐h control and stretch cultures (visualized with anti‐HGF polyclonal antibody). MW‐STD, molecular weight standards (MagicMark Western Protein Standard); PC, recombinant HGF (with BSA of a carrier protein). CNT, control conditioned media from unstretched cells; LFH, LF, and HF, media from stretch cultures ( n = 3 rats each stretch‐pattern group), normalized at constant number of cells after stretch. (e) Activation activity of conditioned media from 1‐h stretch cultures. Conditioned media were collected from control unstretched (CNT) and stretch cultures (LFH, LF, and HF), incubated with 2 μg/mL anti‐HGF neutralizing antibody (AB‐294‐NA from R&D Systems) ( bars b, d, f , and h ) or control IgG ( bar l ) for 30 min at 4°C followed by feeding to unstretched cultures for 24 h beginning 24‐h post‐plating. Experiments included a control set of cultures that were maintained in freshly prepared media (DMEM‐10% HS) with/without 2.5 ng/mL HGF ( bars j–l ). Note that there was no activation activity shown by conditioned medium from HF stretch ( red bar g ) as the BrdU‐incorporation level was comparable to control cultures ( bars a and j ), and was rescued by addition of recombinant HGF to the medium ( red bar i ). Bars represent mean ± SEM, and significant differences from the negative control (CNT) at p < 0.01 are indicated by double asterisks (**) (panels c and e). (f) Visualization of nitrated HGF in HF‐stretch conditioned media by western blotting. Blots were treated with HRP‐labeled anti‐nitrotyrosine mAb (upper row), followed by stripping with SDS‐βME solution and re‐probing with HRP‐labeled anti‐HGF α‐chain mAb (lower row; immuno‐positive α‐chain was indicated by arrowhead). Lanes 1–9 (from left to right): nitrated BSA (positive control), MW‐STD (MagicMark molecular weight standards), recombinant HGF incubated with and without peroxynitrite (ONOO − ), conditioned media from 1‐h LFH‐ and HF‐stretch cultures (2 rats each stretch group), and SDS‐βME sample buffer (SB).

Article Snippet: Interestingly, when the SDS‐βME wash step was omitted, the immuno‐reactivity of the anti‐HGF polyclonal neutralizing antibody (R&D Systems; heavily used in our previous studies) (Elgaabari et al., ; Tatsumi et al., , , ; Tatsumi, Liu, et al., ; Tatsumi, Sankoda, et al., ) drastically decreased with nitration of HGF (Figure , mid column).

Techniques: Labeling, BrdU Incorporation Assay, Activation Assay, Western Blot, Negative Control, Recombinant, Control, Molecular Weight, Activity Assay, Incubation, Stripping Membranes, Positive Control

Journal: Aging Cell

Article Title: Age‐related nitration/dysfunction of myogenic stem cell activator HGF

doi: 10.1111/acel.14041

Figure Lengend Snippet: Peroxynitrite induces nitration/dysfunction of HGF. (a) In vitro experimental design for acute exposure of recombinant HGF to peroxynitrite (panels a–h). Two‐step dilution protocol for the peroxynitrite stock (0.1 M ONOO − on ice) was optimized for the exposure of recombinant HGF (carrier protein‐free) to active peroxynitrite in order to examine whether HGF undergoes nitration and dysfunction under physiological conditions. Lower column is a supplemental sketch of the tyrosine‐residue nitration, peroxynitrite‐induced nitrotyrosine formation by introduction of a nitro group (‐NO 2 ) into a Cε atom in aromatic ring of the side chain, of which surface exposure indexed by the ASA value (see Table ) contributes to high nitration‐selectivity of tyrosine residues. (b) Visualization of HGF nitration status by ECL‐western blotting (peroxynitrite molar ratio dependence). Recombinant mouse HGF (disulfide‐linked heterodimer of 60‐kDa α‐chain and 30‐kDa β‐chain as the major form in the product) was exposed to peroxynitrite in a wide range of molar ratios relative to HGF (1:0–1:4000) at pH 7.2, 37°C for 30 min. Blot was first treated with HRP‐conjugated anti‐nitrotyrosine mAb (upper row), followed by direct re‐probing with anti‐HGF polyclonal antibody (mid row; neutralizing antibody from R&D Systems) and then by re‐probing with the same brand of anti‐HGF polyclonal antibody after stripping with SDS‐βME solution (lower row). Short 50–70 kDa range that covers 60‐kDa HGF α‐chain (including the NK2 segment responsible for c‐met binding) was displayed; panel g shows that the remaining 30‐kDa β‐chain was negative for nitration of tyrosine residues. BSA nitro , nitrated BSA (a positive control for immunodetection of nitrotyrosine); MW‐STD, MagicMark molecular weight standards; lanes a–f , peroxynitrite‐treated HGF (1:100–1:4000), and lanes a′–f′ , control HGF (1:0) incubated in treatment buffer at pH 7.2 without peroxynitrite for 30 min. (c) pH dependence of HGF nitration by peroxynitrite treatment (pH 7.1–7.6, at 1:2000, 37°C for 30 min). Blot was treated with HRP‐labeled anti‐nitrotyrosine antibody, chemically stripped with SDS‐βME, and re‐probed by anti‐HGF antibody as in panel b, lower row. Lanes a–f , peroxynitrite‐treated HGF (pH 7.1–7.6), lanes a′–f′ , the corresponding control HGF. (d) Reaction time dependence of HGF nitration upon peroxynitrite treatment (in a range of 0–300 s; at 1:2000, pH 7.2, 37°C). Blot was treated with HRP‐labeled anti‐nitrotyrosine and anti‐HGF antibodies in the same way as panel c ( lanes a–e ). (e) Densitometry for blots shown in panels b and c. Nitration levels of HGF were normalized by whole HGF (detected by re‐probing with anti‐HGF polyclonal antibody after the stripping step) and expressed as relative to the values of 1:2000 molar ratio (upper row) and pH 7.2 groups (lower row) indicated by red circles. (f) Dysfunction of peroxynitrite‐treated HGF. Satellite cell cultures were maintained in DMEM‐10% HS with peroxynitrite‐treated HGF (3 ng/mL at the final concentration; treated at 1:200–1:4000 of molar ratios, pH 7.2, for 30 min) for 24‐h period beginning 24‐h post‐plating, and pulse‐labeled with BrdU for 2 h to measure BrdU‐positive cell percentages as described in Figure ( orange bars ; see upper column for the experimental scheme). Control HGF, non‐nitrated HGF (1:0; black bar ) in treatment buffer without peroxynitrite treatment. The experiment included two control groups: control set #1 included regular negative‐control culture in DMEM‐10% HS (nega. CNT; far left open bar ) and positive‐control culture with 3.0 ng/mL HGF (posi. CNT; gray bar ); control set #2 is an important set of second control cultures incubated only with a medium for the 1:4000 peroxynitrite treatment (without HGF; open bar a ) and with 3 ng/mL HGF added back to the post‐peroxynitrite treatment (same condition as the 1:4000 treatment; gray bar b ). (g) Immunolocalization of nitration sites in HGF α‐chain. Broad‐range view (20–100 kDa) of western blotting of HGF treated with peroxynitrite (at 1:4000, pH 7.4, 37°C, for 30 min). MW‐STD, MagicMark molecular weight standards; lane a , a blot treated with HRP‐labeled anti‐nitrotyrosine mAb; lane b , re‐probed with anti‐HGF polyclonal antibody after stripping with SDS‐β‐ME; lane c , re‐probed with HRP‐labeled anti‐HGF α‐chain ( N ‐domain) mAb after the second round of chemical stripping. (h) Receptor c‐met binding activities of peroxynitrite‐treated HGF. Evaluated by sandwich ELISA‐like assay on solid‐phase of recombinant c‐met‐Fc chimera (see left photo of the triplicate assay). Optical absorbance 450–540 nm was presented as a relative unit to the control HGF (right column; black bar , 1:0 peroxynitrite treatment served as a positive control). Open bar a , negative control without HGF (nega. CNT); orange bars , HGF treated with peroxynitrite at 1:4000, 1:2000, and 1:100 at pH 7.2 for 30 min (from left to right). (i) c‐Met binding activities of Y198‐peptide (left column; 7.5 μM FTSNPEVR nitro/non‐nitro Y 198 EV) and Y250‐peptide of HGF (right column; 0.15 μM KFLPER nitro/non‐nitro Y 250 PDKGFD), evaluated by the sandwich ELISA‐like assay (triplicate assay). Optical absorbance was presented as a relative unit to each non‐nitrated peptide ( black bars , served as positive controls). Open bars a , negative controls without peptides (nega. CNT); orange bars , nitroY198‐ and Y250‐peptides. Bars represent mean ± SEM and significant differences from the control HGF ( black bar ) in panel f and the negative control ( bar a ) in panels h and i, at p < 0.05, p < 0.01, and p < 0.001 are indicated by (*), (**), and (***), respectively. (j) Nitration status of HGF α‐chain in response to SIN‐1. Visualized by western blotting of HGF treated with SIN‐1, a chronic generator of peroxynitrite, at 1:0, 1:1000, 1:5000, and 1:10,000 of molar ratios to HGF, pH 7.4, 37°C for 30 min. Blots were treated with HRP‐labeled anti‐nitrotyrosine antibody followed by stripping with SDS‐βME and re‐probing with HRP‐labeled anti‐HGF α‐chain mAb.

Article Snippet: Interestingly, when the SDS‐βME wash step was omitted, the immuno‐reactivity of the anti‐HGF polyclonal neutralizing antibody (R&D Systems; heavily used in our previous studies) (Elgaabari et al., ; Tatsumi et al., , , ; Tatsumi, Liu, et al., ; Tatsumi, Sankoda, et al., ) drastically decreased with nitration of HGF (Figure , mid column).

Techniques: Nitration, In Vitro, Recombinant, Residue, Western Blot, Stripping Membranes, Binding Assay, Positive Control, Immunodetection, Molecular Weight, Control, Incubation, Labeling, Concentration Assay, Negative Control, Sandwich ELISA