Anti ppm1a

The cells were incubated with an appropriate volume of immunoprecipitation lysis buffer (Pierce, Thermo Scientific) and a protease inhibitor mixture for 4 hours at 4°C. The supernatant lysates were collected and were then incubated with 2 μg of primary antibodies overnight at 4°C: anti-Flag (1:100; Proteintech, 66008-3), anti-HA (1:100; Proteintech, 51064-2), anti-NDRG2 (1:100; Abcam, ab174850), and anti-PPM1A (1:100; Cell Signaling Technology, 3549). After incubation for 4 hours at 4°C, the protein A/G magnetic beads (MedChemExpress, HY-K0202) were washed three times with the lysis buffer. The proteins were eluted in loading buffer and analyzed by immunoblotting analysis.

Expression plasmids encoding Flag-, Myc-, or HA-tagged human TBK1, IKKε, IRF3, caRIG-I, MAVS, STING, PPM1A or its D239N and R174G mutations, and the IRF3/7-responsive reporters IFNβ_Luc and 5xISRE_Luc have been described by Lin et al. (28 (link)) and Xu et al. (56 (link)). Site-directed mutagenesis was used to generate expression plasmids encoding TBK1/IKKε with amino acids Ser¹⁷² replaced by Ala or Glu. TBK1 truncations, including TBK1 amino acids 1 to 299, 1 to 382, and 299 to 729 were generated by PCR-based cloning, which was performed using a kit from Stratagene. Detailed information will be provided upon request. All coding sequences were verified by DNA sequencing.
Poly(I:C) was from Invivogen. GFP- and luciferase-tagged herpes simplex virus–1 (HSV-1) was a gift from J. Han (Xiamen University, Xiamen). gVSV was a gift from Z. J. Chen (University of Texas Southwestern Medical Center, Dallas). SeV (Cantell strain) was from Charles River Laboratories. λPPase was from BioLabs. The monoclonal anti-IRF3, anti-pIRF3, anti-TBK1, anti-pTBK1 (S172), anti-IKKε, anti-IKKε (S172), anti-MAVS, anti-Myc, anti-PPM1A, and anti-HA antibodies were from Cell Signaling Technology. Anti–α-tubulin and anti-Flag antibodies were from Sigma. Ppm1a^−/− C57BL/6 mice were generated by Feng Laboratories.