ERK1/2 phosphorylation assay

Agonist-induced ERK1/2 phosphorylation was measured primarily using a previously described Western blotting–based protocol (33). C5aR1-, C5a-V2R–, or C5aR2-expressing stable cell lines were seeded into 6-well plate at a density of 1 million cells/well. The cells were serum-starved for 12 h followed by stimulation with 1 μm of C5a and 10 μm of C5a^pep, respectively, at selected time points. After the completion of time course, the medium was aspirated, and the cells were lysed in 100 μl of 2× SDS dye/well. The cells were heated at 95 °C for 15 min followed by centrifugation at 15,000 rpm for 10 min. 10 μl of lysate was loaded per well and separated on SDS-PAGE followed by Western blotting. The blots were blocked in 5% BSA (in TBST) for 1 h and incubated overnight with rabbit phospho-ERK (catalog no. 9101/CST) primary antibody at 1:5000 dilution. The blots were washed thrice with TBST for 10 min each and incubated with anti-rabbit HRP-coupled secondary antibody (1:10,000, catalog no. A00098/Genscript) for 1 h. The blots were washed again with TBST for three times and developed with Promega ECL solution on chemidoc (Bio-Rad). The blots were stripped with low pH stripping buffer and then reprobed for total ERK using rabbit total ERK (catalog no. 9102/CST) primary antibody at 1:5000 dilution. To measure the effect of PTX on ERK activation, HEK-293 cells stably expressing C5aR1 were seeded in 6-well plate. The cells were treated with 100 ng/ml PTX for 12–16 h followed by serum starvation. The cells were stimulated with C5a and C5a^pep for the indicated time points. The samples were prepared and processed as mentioned previously.

The ligand-induced phospho-ERK1/2 signaling in HMDMs was assessed using the AlphaLISA Surefire Ultra p-ERK1/2 (Thr²⁰²/Tyr²⁰⁴) kit (PerkinElmer) following the manufacturer's protocol. Briefly, HMDMs were seeded (50,000/well) in tissue culture-treated 96-well plates (Corning) for 24 h and serum-starved overnight. All ligand dilutions were prepared in serum-free medium containing 0.1% BSA. For stimulation, the cells were incubated with respective ligands for 10 min at room temperature and then immediately lysed using AlphaLISA lysis buffer on a microplate shaker (450 rpm, 10 min). For the detection of phospho-ERK1/2 content, cell lysate (5 μl/well) was transferred to a 384-well ProxiPlate (PerkinElmer) and added to the donor and acceptor reaction mix (2.5 μl/well, respectively) with 2-h incubation at room temperature in the dark. On a Tecan Spark 20M, following laser irradiation of donor beads at 680 nm, the chemiluminescence of acceptor beads at 615 nm was recorded. To measure the effect of PTX, culture medium was changed into serum-free IMDM containing 200 ng/ml PTX or vehicle only for overnight incubation. Afterward, ERK1/2 phosphorylation was measured as described above.