Hrp secondary antibody

ATN-291 was manufactured by SDIX [12 (link)]. Fluorescein isothiocyanate (FITC)- and Cy3-labeled secondary antibodies used in flow cytometry or histology were purchased from Jackson Immunoresearch Laboratories, Inc. (West Grove, CA). Anti-uPA antibody, anti-uPAR antibody and anti-β-actin antibody (conjugated with horseradish peroxidase [HRP]) used in Western blotting were both purchased from Abcam (Cambridge, MA, USA). Secondary HRP antibodies were purchased from Jackson ImmunoResearch (St. Louis, MO, USA). p-SCN-Bn-Df (i.e. p-isothiocyanatobenzyl-desferrioxamine B) was acquired from Macrocyclics, Inc. (Cat #: B-705, Dallas, TX). Chelex 100 resin (50 – 100 mesh) was purchased from Sigma-Aldrich (St. Louis, MO). Buffers used in this study were prepared from Millipore-grade water and pre-treated with Chelex 100 resin to ensure that the aqueous solution was free of heavy metals. Size exclusion PD-10 columns were purchased from GE Healthcare (Piscataway, NJ). All other chemicals were purchased from Thermo Fisher Scientific (Fair Lawn, NJ).

Enriched splenic DCs were cultured in 96- or 24-well plates with purified flagellin (1 μg/mL or 10 ng/mL) and lysed at various time points with RIPA buffer (Sigma) containing protease inhibitors (Sigma). Whole-cell extracts were centrifuged, loaded onto 4–15% Tris/glycine gels (Bio-Rad, USA), and transferred to polyvinylidene fluoride or nitrocellulose membranes (PVDF)(GE Healthcare, Pittsburgh, PA, and Invitrogen). All antibodies (anti-phospho PKC-delta, anti-caspase-1, anti-β-actin, anti–TEK, and anti-phospho-IRS-1) were added at 1:1000 dilution, followed by HRP secondary antibodies (Jackson Immuno Research, West Grove, PA). ECL plus (GE Healthcare) was used as substrate and blots examined using a Kodak Image Station.

Cardiomyocytes were dissociated by TrypLE around day 10, and different doses of nicotinamide or ROCK inhibitor Y27632 10 μM were added in the medium for 1 h. The protein was harvested after 1 h for western blot [22 ]. Briefly, 40 μg protein of each sample was separated by electrophoresis with SDS-PAGE gels, and then transferred to PVDF membranes. The membranes were first blocked with 5% non-fat milk, and then incubated with antibodies against p-MLC (Ser19) 1:500 (Cell Signaling, 3671), MLC 1:1000 (Sigma, M4401), or Actin 1:2000 (Santa Cruz, sc-47778) overnight at 4 °C. After washing, the membranes were incubated with HRP-secondary antibodies (Jackson, 115-035-146 or 111-035-144) for 2 h at room temperature. Chemiluminescence was detected using SuperSignal^TM West Pico PLUS Chemiluminescent Substrate (Thermo) or West Dura Extended Duration Substrate (Thermo).

For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose. Lysates were separated by SDS–PAGE on 8% Tris‐glycine gels (8% [w/v] acrylamide, 0.4 M Tris–HCl pH 8.8 0.08% [w/v] SDS, 6.2% [v/v] glycerol 0.05% [v/v] temed, and 0.02% [w/v] APS). Gels were resolved for 90 min at 35 mA and transferred to 0.2 μm nitrocellulose membrane (Hybind‐C, GE Healthcare, Amersham, UK) for 2 hr at 125 mA. Membranes were blocked in 5% (w/v) bovine serum albumin (BSA) diluted in Tris‐buffered saline (TBS) with 0.1% (v/v) Tween‐20 (TBST) and probed with primary antibodies, directed against: ERK5 (#3372), phospho ERK1/2 (#4370), GAPDH (#5174), phospho ERK5 (T218/Y220) (#3371), ERK1/2 (#4695) were purchased from New England Biolabs (Hitchin, UK). Antibodies to RAP1 (SC‐65) and unprenylated RAP1A (SC‐1482) were purchased from Santa Cruz Biotechnology. Antibodies to MEKK2 (ab33918), MEKK3 (ab40750), and MEK5 (ab45146) were purchased from Abcam (Cambridge, UK). Antibody to ZO‐1 (40‐2200) was purchased from Invitrogen (Thermo Fisher Scientific). All antibodies were diluted in 1% (w/v) BSA and incubated overnight at 4°C. Proteins were detected using rabbit‐ or goat‐specific HRP secondary antibodies (Jackson Immunoresearch Labs) and enhanced‐chemiluminescence (ECL) Western blotting detection reagent (Pierce).