Igg1 isotype control

For TNF-α blocking experiments, 5 μg/mL TNF-α neutralizing antibody (monoclonal rat IgG1, clone MP6-XT22) or IgG1 isotype control (both from R&D Systems) was added to MLO-Y4 cells for 1 h prior to the addition of RAW264.7 macrophage conditioned medium for 24 h. The concentration of TNF-α neutralizing antibody was chosen based on optimization experiments whereby three different concentrations (0.5, 2.5, and 5 μg/mL) of neutralizing antibody was added to MLO-Y4 cells prior to the addition of 4 ng/mL TNF-α for 24 h. The addition of 5 μg/mL TNF-α neutralizing antibody suppressed TNF-α-induced expression of COX-2, IL-11, and RANKL genes by MLO-Y4 cells (data not shown).
For COX-2 blocking experiments, 1 μM COX-2-specific inhibitor (SC-236, Sigma-Aldrich) was added to MLO-Y4 cells for 1 h prior to the addition of RAW264.7 macrophage conditioned medium for 24 h.

In a transwell co-culture system, RAW 264.7 cells (2×10⁵, 4×10⁵, 8×10⁵) seeded on a 0.4 μm Transwell insert (Millipore) were co-cultured with podocytes (4×10⁵) for 48 h in the absence or presence of 25 mM high glucose treatment. The ratios of podocytes (P) to macrophages (M) were 2:1, 2:2 and 2:4, respectively.
In the CM experiments, podocytes (4×10⁵) planted on six well plates were cultured overnight in normal RPMI 1640 media. Then, the cells were washed with PBS three times. After that, normal PRMI 1640 media (control), NC-CM or HG-CMwas added to podocytes for 24 - 72 h. In some experiments, 10μg/ml anti-TNF-α neutralizing antibody (RD, USA), 10μg/ml IgG1 Isotype control (RD, USA), 300μM ROS inhibitors (Tempo, sigma) or 10μM p38MAPK inhibitor (SB203580, RD, USA) was respectively added to cells with CM for 72 h. Besides, 10ng/ml recombinant mouse TNF-α (RD, USA) alone was applied to incubate podocytes for 72 h when necessary.

The following reagents were from Sigma–Aldrich (St. Louis, MO, USA); 16-mercaptohexadecanoic acid, cysteamine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, succinic anhydride and N-hydroxysuccinimide. Triethylamine, dimethylformamide and bovine serum albumin (BSA) were from Fisher Scientific (Pittsburgh, PA, USA). Six-armed-poly(ethyleneglycol)–amine was from SunBio (South Korea). PBS (Cat#70011-036) and Tween-20 were from Life Technologies (Grand Island, NY, USA).
Sandwich antibody pairs for IL-20 were produced by Novo Nordisk A/S. Capture anti-IL-20 antibody was of the IgG1 isotype. Unspecific binding was assessed using a IgG1 isotype control from R&D Systems (MAB002, Minneapolis, MN, USA). IRDye-800 streptavidin conjugate was from LiCor Biosciences (Lincoln, NE, USA). Mouse, goat and bovine IgG were from Jackson ImmunoResearch (West Grove, PA, USA).
Purified cytokine antigen standards and sandwich antibody pairs for IL-1β (MAB601, BAF201), IL-10 (MAB2172, BAF217), IL-6 (MAB206, BAF206), and TNFα (MAB610, BAF210) were purchased from R&D systems. All capture antibodies were of the IgG1 isotype.

Samples (cells or tissues) were washed twice with PBS and fixed in ice-cold acetone for 5 min at room temperature, air dried, rehydrated in PBS and then blocked for 1 h in Dako Serum-Free Protein Block (DSFPB) (Agilent Technologies, Santa Clara, CA). Samples were incubated with purified primary antibodies (or suitable isotype matched controls) in DSFPB for 2 h at room temperature, washed in PBS, and then probed with Alexa-Fluor 647 labeled anti-Murine IgG raised in goat (Thermo Fisher Scientific) for 30 min. Following washing in PBS, samples were stained with fluorophore-conjugated primary antibodies for 2 h at room temperature if required. Samples were washed in PBS, counterstained with DAPI for 10 min and mounted with a coverslip using Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Immunofluorescence images were acquired using a Zeiss Cell Observer SD confocal fluorescence microscope (Zeiss, Oberkochen, Germany). Fluorochrome-labeled antibodies: AF594 anti-CD31 (catalogue number 303126; BioLegend, San Diego, CA), AF647 anti-mouse IgG raised in goat (catalogue number A21235; Thermo Fisher Scientific). Unconjugated antibodies for IFM: anti-ICAM-1, anti-VCAM-1, anti-P-selectin and IgG1 isotype control (catalogue numbers BBA3, BBA5, BBA30, MAB002 respectively; R&D, Abingdon, UK).

Surface expression of the VEGFR1 on CD14⁺⁺CD16^‐ monocytes was determined by flow cytometric analysis using a FACSCalibur (BD). 0.5 × 10⁶ cells were stained with 0.5 µg Anti‐human VEFR1/Flt‐1‐Phycoerythrin or the corresponding IgG1 isotype control (both R&D system) and used directly for FACS analysis. Events are defined in the forward scatter versus side scatter plot, and afterwards, a gate was defined around the cells. The mean of all stained cells was identified in a single coloured histogram (FACSDiva Version 6.1.3 Software). The relative fluorescence intensity (RFI) was calculated by dividing the mean of VEGFR1 stained cells with the mean of isotype‐stained cells from all samples and used for further analysis.