Mouse monoclonal anti flag m2 affinity gel

The full-length open reading frame of the human PLD3 gene or the human GRN gene, selected as a candidate for PLD3 interactors, was amplified by PCR using PfuTurbo DNA polymerase (Agilent Technologies, Palo Alto, CA, USA) and the set of sense and antisense primers. Subsequently, PCR products were cloned in the expression vector named p3XFLAG-CMV7.1 (Sigma), pCMV-Myc (Clontech, Mountain View, CA, USA), pDsRed-Express-C1 (Clontech), or pZsGreen1-N1 (Clontech) to express a fusion protein with an N-terminal Flag tag, Myc tag, DsRed tag, or C-terminal ZsGreen tag, respectively. The vectors were transfected in HEK293 cells, HeLa cells or SK-N-SH cells using Lipofectamine 2000 reagent (Invitrogen) for transient expression. The M232 isoform of PLD3 was prepared from the cloned vector using QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). After cotransfection of the vectors, the protein extract was processed for immunoprecipitation with mouse monoclonal anti-Flag M2 affinity gel (Sigma) or rabbit polyclonal anti-Myc-conjugated agarose (Sigma), followed by western blot with a rabbit polyclonal anti-Myc antibody (Sigma) or a mouse monoclonal anti-FLAG M2 antibody (Sigma), respectively.

Cisplatin (S1166), Adriamycin (S1208), Hydroxyurea (S1896), Rucaparib (S1098), 5-Fluorouracil (S1209), DMXAA (S1537), BFA (S7046), Rapamycin (S1039), and 3-MA (S2767) were from Selleck. Doxycycline hyclate (D9891), Phenylarsine oxide (P3075), GTP-Agarose suspension (G9768), Protein A agarose (P3476), mouse monoclonal Anti-Flag M2 Affinity Gel (A2220), and mouse monoclonal Anti-HA-Agarose antibody (A2095) were from Sigma-Aldrich. DiABZI STING agonist-1 (Tautomerism) (HY-112921) and Digitonin (HY-N4000) were from MCE. HaloTag® Ligands (GA1110) for super-resolution microscopy were from Promega. 2'3'-cGAMP (tlrl-nacga23-1) was from Invivogen. Pierce™ Anti-DYKDDDDK Magnetic Agarose (A36797) was from Thermo Fisher Scientific.