Casein solution

Neutrophils were recruited in BALB/c or C57BL/6 mice 3 h after intraperitoneal injection of 1 ml of 9% casein solution (Sigma). Mice were euthanized and the peritoneal cavity was washed with RPMI 1640 (Sigma) without serum at room temperature. A fraction of the obtained cells was first characterized by flow cytometry using anti-Ly6G-PerCP (murine, eBioscience) revealing >95% Ly6G⁺ cells (neutrophils). Remaining cells were centrifuged at 400 g for 5 min, resuspended in RPMI and used in the following assays.

Alginic acid, CaCl₂, casein solution, DPX mountant, eosin, EDTA, EtOH, haematoxylin, NaCl, paraformaldehyde (PFA), polyvinylpyrrolidone (PVP), sodium citrate, Triton™ X-100 and xylene substitute were purchased from Sigma-Aldrich (Gillingham, UK). Hydroxypropyl-methylcellulose (HPMC) was purchased from Alfa Aesar (Thermo Fisher Scientific, Heysham, UK).

Proteins were separated by 8% or 10% SDS–PAGE and transferred onto PVDF membranes (0.45 mM, #T830.1; Carl Roth). The membranes were blocked in 5% skimmed milk powder (Sigma-Aldrich) or in casein solution (Sigma-Aldrich) for 1 h at RT. Primary antibodies were incubated overnight at 4°C in 5% skimmed milk and 0.1% Tween. Membranes were washed 3× in TBS/0.05% Tween and incubated with secondary antibody for 1 h at RT. Membranes were washed 3× in TBS/0.05% Tween. Proteins were detected either after short incubation in Immobilon Western ECL substrate (#WBKLS0500; Millipore) on an Odyssey FC device (Li-cor Biosciences) or on an Odyssey CLx scanner (Li-cor Biosciences). The following antibodies were used: ADAM19_74–123 (#NBP1-69367; Novus), ADAM19_218–267 (#ab104800; Abcam), FLAG (#TA50011-100; Origin), PTHR1_4–54 (#ABIN2776779; Antibodies-Online.de), PTHR1_52–86 (#ABIN5708269; Antibodies-Online.de), FLAG (#TA50011-100; Origin), HA (#137838; Abcam), polyclonal PTHR1_573–593 (Lupp et al, 2010 (link)), anti-mouse IgG-HRP (#P0260; Dako), anti-rabbit IrDye680 (#926-32223; Li-cor Biosciences).