Anti il 10 ab9969

The method for protein from total kidney homogenate was extracted according to the manufacturer’s recommended protocol (Cwbiotech, Beijing, China). Proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (GE Healthcare, Freiburg, Germany). After transfer, 5% skimmed milk powder was used to block the membranes at room temperature for 1 h. Then, the membranes were pre-incubated with primary Foxp3 (sc-53,876, 1:500 dilution), IL-6 (sc-7920, 1:500 dilution) (Santa Cruz Biotechnology, California, USA), anti-IL-10 (ab9969, 1.5 μg/mL dilution), anti-IL-17 (ab79056, 1.5 μg/mL dilution) (Abcam Ltd., Hongkong, China) and GAPDH (1:1000 dilution) (Danvers, MA, USA) antibodies at 4 °C overnight and then incubated with secondary antibodies following three washes with TBST. Then, to detection of the expression level of the protein of interest using an enhanced chemiluminescence (ECL, Millipore, Billerica, MA, USA) western blot detection kit and a chemiluminescent imaging system (Bio-Rad, USA)..

For Western-Blot sampling, sorted cells from 5 animals were pooled. The cell pellet was lysed with 50 μL of ice-cold RIPA buffer. Thereafter, Laemmli Sample Buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was added with heating at 95 °C. The proteins were separated by 10%–12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a membrane as described previously [57 (link)]. After blocking membranes were stained overnight with antibodies to Arg1, iNOs, CCR7, IL6, IL10, FXYD2, ALOX12, or GAPDH (anti-Arginase I: sc-18351, 1:200; anti-NOS2:sc-651, anti-GAPDH:sc-25778, Santa Cruz, USA, anti-CCR7:ab32527, anti-IL6:ab7737, anti-IL10:ab9969, Abcam, UK), anti-FXYD2:PA5-75640, anti-ALOX12:PA5-78760, ThermoScientific, Waltham, MA, USA), washed thrice, and stained with HRP-conjugated antibodies (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Bands were imaged with a Novex ECL Kit (Invitrogen, Carlsbad, CA, USA) in a ChemiDoc™ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Densitometry analysis was carried out using ImageLab Software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH was used as a reference protein. Uncropped membranes after blotting are available in Supplementary Materials.