Pab18350

Antigen retrieval was performed using 20–40 µg/ml proteinase K (Sigma) digestion at 37°C for 15 min. The sections were then blocked with 10% goat serum and 0.5% bovine serum albumin (BSA) in 3% milk for 1 h at room temperature, followed by primary antibody labeling, mouse–antimouse F4/80 (1:100, ab100790, Abcam) or rabbit–antimouse erythropoietin receptor (EPOR, 1:800, PAB18350, Abnova) at 4°C overnight. The next day, DAKO secondary (K4063) was applied to the sections for 30 min. The antibody binding was then revealed by 3,3′-diaminobenzidine (DAB, Vector, Burlingame, USA) and hematoxylin counterstaining.
For double labeling of properdin and F4/80 in the kidneys of WT mice, proteinase K at 40 µg/ml was used for antigen retrieval at 37°C for 30 min. After the staining of F4/80 was obtained, the sections were then incubated with primary antibody rabbit–antimouse properdin (1:200, ABF185, Merck Millipore) at 4°C overnight. Afterwards, biotinylated secondary goat–antirabbit immunoglobulin G (IgG) was applied to the slides for 30 min at 37°C (1:300, BA-1000, Vector), followed by alkaline phosphatase streptavidin (1:200, SA-5100, Vector) for 30 min at 37°C, and then developed by Fast Red (Sigma) for 8 min.

Twenty-five micrograms of protein was separated by the electrophoresis of polyacrylamide denaturing gels and transferred onto polyvinylidene fluoride membrane. The primary antibodies, rabbit–antimouse caspase-3 (1:400, 9662, CST, Danvers, USA), rabbit–antimouse high-mobility group box-1 protein (HMGB1, 1:1,000, 3935, CST), mouse–antimouse proliferating cell nuclear antigen (PCNA, 1:1,000, M0879, DAKO, Glostrup, Denmark), rabbit–antimouse erythropoietin receptor (EPOR, 1:1,000, PAB18350, Abnova, Taiwan), rabbit–antimouse properdin (1:1,000, AB186834, Abcam, Cambridge, USA) and mouse–antimouse β-actin (1:5,000, A5441, Sigma, Dorset, UK), were applied to the membranes overnight at 4°C, followed by the incubation of horseradish peroxidase-labeled secondary antibody (goat–antirabbit/mouse, K4063, DAKO) and developed by enhanced chemiluminescence (Thermo Fisher Scientific, Rockford, USA). The ratio of target protein to β-actin in volume density, as an endogenous loading control, was calculated for each detection, and then, the fold change of detected protein in the experimental group against the WT sham control was obtained as final results (27 (link)).

The EPOR protein was immunoprecipitated from 200 μg tissue/cell homogenates through incubation with 1 μg of anti-EPOR antibody (PAB18350, Abnova) for overnight at 4 °C on a rotator. Then, 40 μl protein A sepharose beads (17-0469-01, GE healthcare, Pittsburgh, USA) was added to each sample and incubated for another 2 h at 4 °C on the rotator. Afterwards, the beads were collected by spinning at 500 g for 30 s and washed 3 times with RIPA buffer. The supernatant was discarded and 25 μl of 4×loading buffer (Bio-Rad) was added to each sample and boiled for 10 min at 100 °C on a heat block. The samples were separated by SDS-PAGE gels, transferred onto PVDF membranes and probed with anti-βcR antibody (sc-93281, Santa Cruz, Dallas, USA). The final detected βcR bands represent the level of EPOR/βcR heterodimer.