Pierce crosslink magnetic ip co ip kit

Co-IP was conducted using Pierce Crosslink Magnetic IP/Co-IP Kit in accordance with the manufacturer’s instructions (Thermo Fisher Scientific, CA, USA). Briefly, cell lysates were incubated with protein A/G magnetic beads which were previously bind to primary antibodies, then dissociated the bound antigens from antibody-crosslinked beads by eluting in a low-pH elution buffer. The eluent was detected by WB analysis.

Immunoprecipitations from whole kidney lysates were carried out by using the Pierce Crosslink Magnetic IP/Co-IP Kit from Thermo Scientific, according to manufacturer’s instructions, and from cell lysates as previously described24 (link). A detailed description is provided in the Supplementary Methods.

Cells or tissues were lysed in immunoprecipitation lysis buffer provided in Pierce™ Crosslink Magnetic IP/Co-IP Kit (Thermo Fisher) supplemented with 5 mM of nicotinamide, 5 μM of trichostatin A, protease, and phosphatase inhibitors for 30 min on ice. Lysate was incubated with the indicated antibodies cross-linked to Protein A/G magnetic beads for 1 h at room temperature before washing three times in immunoprecipitation lysis buffer and then eluted.

Co‐IP assays of DEPDC1B and Rac1 were performed according to the manufacturer's instructions of the Pierce Crosslink Magnetic IP/Co‐IP Kit (88805, Thermo Scientific). Briefly, 2 µg of an anti‐Flag (14793), anti‐IgG(3900, CST), or anti‐Rac1(ab33186, Abcam) antibody was bound to Protein A/G Magnetic Beads, and the antibody was covalently cross‐linked to the beads using disuccinimidyl suberate (DSS). The antibody cross‐linked beads were then incubated overnight at 4°C with 400 µg cell lysate that contained Flag‐DEPDC1B and Rac1. The beads were washed to remove unbound proteins, and a low pH elution buffer was used to dissociate bound antigen from the antibody cross‐linked beads. Neutralization buffer was included to prevent precipitation of the isolated antigen and to ensure protein activity in downstream applications. Lane Marker Sample Buffer was used to prepare samples for SDS‐PAGE immunoblot analysis with specific antibodies against or the SDS‐PAGE gel was silver stained by using the Pierce Silver Stain Kit (24612, Thermo Scientific). The proteins eluted from magnetic beads were followed by mass spectrometry (MS) analysis with an Easy nanoLC 1200‐Orbitrap Fusion (Thermo Fisher, USA), and the proteins identified by MS were analyzed on the Metascape website (http://metascape.org) and the String website (https://string-db.org).

Total proteins were used for immunoprecipitation using Pierce™ Crosslink Magnetic IP/Co‐IP Kits (#88805, Thermo Scientific Pierce™ Crosslink Magnetic IP/Co‐IP Kit, Thermo Fisher Scientific, IL, USA) as recommended by the supplier. Primary antibodies against Bmi‐1 (#5856, Cell Signaling Technology, USA), RING1B (#5694, Cell Signaling Technology, USA), GATA4 (#19530, Proteintech, USA), p62 (#39749, Cell Signaling Technology, USA), anti‐ubiquitin (#91112, Cell Signaling Technology, USA), HSC70 (10654‐1‐AP, Proteintech, USA), DYKDDDDK Tag (Anti‐FLAG M2 antibody, #14793, Cell Signaling Technology, USA), His‐Tag (#12698, Cell Signaling Technology, USA), HA‐Tag (#3724, Cell Signaling Technology, USA) and Myc‐Tag (M1405‐5, Hangzhou HuaAn Biotechnology, China) were used.

Antibodies information can be found in Supplementary Table S1. Lipofectamine® 3000, RNAiMAX, MessengerMAX, Opti-MEM® reduced serum media, and SYBR® Select Master Mix, were obtained from Invitrogen® (Thermo Fisher, Life-Technologies, Carlsbad, CA, USA). RPMI-1640, DMEM medium, and FBS were obtained from Gibco® (Thermo Fisher, Life-Technologies, Carlsbad, CA, USA). PrimeScript® RT reagent Kit with gDNA Eraser was obtained from Takara bio (Dalian, China). TGF-Beta1 was purchased from PeproTech® (London, UK). mMESSAGE mMACHINE® T7 ULTRA Transcription Kit was obtained from Ambion® (Thermo Fisher, Life-Technologies, Carlsbad, CA, USA) LY2109761(Cat.#S2704) was obtained from Selleck (Houston, TX, USA)
SimpleChIP® Plus Enzymatic Chromatin IP Kit-Magnetic Beads (#9005) were purchased from Cell Signaling Technology (Danvers, MA, USA), and Pierce® Crosslink Magnetic IP/Co-IP Kit(#88805) came from Thermo Fisher Scientific (Waltham, MA, USA). siRNAs for RbBP5, SNAI1, CBP, SMAD2 were synthesized by Ribo Bio(Guangzhou, China). For their sequences, see the Supplementary Table S2.