Pierce anti dykddddk magnetic agarose

Antibodies against IMUP (1:500; #ab228823), pCDC25A (Phospho S124; 1:1000; #ab156574), pCHK1 (1:500; ab79758), from Abcam; cyclin-dependent kinase 2 (CDK2) (1:1000; #AF6237) from Affinity Biosciences (OH, USA); Cyclin A2 (1:1000; #BF683) and Cyclin E1 (1:1000; #HE12) from Cell Signaling Technology (Danvers, MA, USA); and FHL1 (1:500; #10,991–1-AP), CDC25A (1:1000; #55,031–1-AP), CHK1 (1:1000; #25,887–1-AP), CDC25C (1:1000; #16,485–1-AP), 14–3-3ξ (1:1000; #11,648–2-AP), SP1 (1:1000; #21,962–1-AP), and NPM1 (1:1000; #10,306–1-AP) from Proteintech were used for WB assays.
Antibodies against IMUP (1:100; #ab228821) from Abcam; GFP-Trap® Magnetic Agarose (20 μL per reaction; #gtma-100) from Proteintech; and Pierce™ anti-DYKDDDDK Magnetic Agarose (20 μL per reaction; #A36798) from Invitrogen were used for IP to detect protein interactions. For LC/MS, immunocomplexes from GFP-Trap® Magnetic Agarose, anti-IMUP agarose, or anti-DYKDDDDK magnetic agarose were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Subsequently, the candidate bands were cut and identified by LC/MS.

RNA immunoprecipitation (RIP) analysis was performed according to the protocol provided by Medical and Biological Laboratories Co. Ltd. (MBL, Bei Jing, China) and Ribocluster Profiler RIP-Assay kit (catalog RN1001, MBL). Flag-PTRF overexpression cells were collected in 1.5 mL centrifuge tubes from the 100 mm culture dish using a cell scraper. We prepared 10 million cells per sample, and each sample had two biological replicates. Next, cells were washed twice with nuclease-free ice-cold PBS. The cell suspension was centrifuged at 300× g for 5 min at 4 °C. After removing the supernatant, 500 μL of lysis buffer(+ 1mM Dithiothreitol) was added to each sample, and the mixture was vortexed thoroughly. The sample was incubated for 10 min on ice and centrifuged at 12000× g for 10 min at 4 °C. The cell pellet was removed, and the supernatant was transferred to the tube containing Pierce™ Anti-DYKDDDDK Magnetic Agarose (Invitrogen, A36798, Carlsbad, State of California, America), and then washed once with the lysis buffer (+ 1 mM Dithiothreitol). The tube was incubated with rotation overnight at 4 °C. The tube was placed on a magnetic stand, and the supernatant was removed. Beads were used to isolate and purify the RNA. Purified RNA was used for reverse transcription and the library construction of high-throughput sequencing. RIP experiments were performed in two biological replicates.

To generate and purify Flag-TAZ/Myc-USP7-WT recombined proteins from mammal cells, HEK293T cells were transfected with the indicate plasmid and cultured in SFM4Transfx-293 without L-Glutamine culture medium (SH30860.02, Thermo) for another 4 days. Cells were harvested and the recombined proteins were purified by using Pierce™ anti- DYKDDDDK Magnetic Agarose (A36797, Thermo)/ Pierce™ Anti-c-Myc Agarose (20168, Thermo) and then eluted with Pierce™ 3×DYKDDDDK peptides (A36805, Thermo)/Pierce™ c-Myc Peptide (20170, Thermo). The purified proteins were verified by SDS-PAGE.
The purified Flag-TAZ and Myc-USP7-WT were mixed with equimolar, incubated (supplemented with protease inhibitor mixture and Pierce™ anti- DYKDDDDK Magnetic Agarose) at 4 °C overnight, and followed by 5-time IP lysis buffer washing step. The magnetic agarose was resuspended and routinely subjected to SDS-PAGE process.