Anti p ser

The Escherichia coli 0111: B4 LPS, N-hexanoyl-D-sphingosine (C6-ceramide) and zVAD-FMK were obtained from Sigma-Aldrich (St. Louis, MO, USA). DAPK1 inhibitor and ST2825 were purchased from Medchem Express (Monmouth Junction, New Jersey, USA). Recombinant GST-DAPK1 fusion protein was obtained from Millipore (Billerica, MA). Caspase-3 activity detection kit was from Bestbio (Shanghai, China). Quantikine human IL-6 ELISA kit was from R&D Systems (Minneapolis, MN). Annexin V-FITC apoptosis detection kit was ordered from Beyotime (Nanjing, China). The following antibodies with the company and concentration were used for coimmunoprecipitation (co-IP) or western-blotting analyses: anti-Pellino1 (Abcam, 1:500), anti-MyD88 (Cell Signaling Technology, 1:500), anti-caspase-8 (Cell Signaling Technology, 1:500), anti-TRIF (Cell Signaling Technology, 1:1000), anti-RIP1 (BD Biosciences, 1:2000), anti-Flag (ProteinTech group, 1:1000), anti-phospho-DAPK1 (Sigma-Aldrich, 1:1000), anti-DAPK1 (Cell Signaling Technology, 1:1000), anti-Fbxw7 (Abcam, 1:1000), anti-pSer (Santa Cruz, 1:1000), anti-Fn14 (Cell Signaling Technology, 1:2000) and anti-GAPDH (Biosynthesis, 1:3000).

Polyclonal anti-T. gondii antibody was raised in New Zealand rabbits by several injections of 50 µg of soluble T. gondii antigen suspended in Freund’s incomplete adjuvant. The IgG fraction of this serum was purified by chromatography on DEAE Trisacryl (l M) and tested by ELISA. The mouse monoclonal antibody against UHRF1 (clone 1RC1C-10) was engineered as described elsewhere [28 (link)]. The anti-cyclin B1, anti-p-Ser and anti-actin mouse monoclonal antibodies or anti-DNMT1 rabbit polyclonal antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Alexa Fluor 488 goat anti-rabbit IgG was obtained from Invitrogen (Carlsbad, CA).
Goat F(ab’)2 fragment anti-mouse IgG-peroxidase, donkey F(ab’)2 fragment anti-rabbit IgG-peroxidase, Tween-20 and the protease inhibitor cocktail were purchased from Roche Diagnostics (Basel, Switzerland). G 418 was purchased from Sigma-Aldrich (Saint-Louis, MO).
The ECL detection system was obtained from Amersham Biosciences (GE Healthcare Europe GmbH, Orsay, France). TriReagent was purchased from Molecular Research Center (Cincinnati, OH).

Cells were lysed in PBS containing 1 mM Na₃VO₄, 1 mM NaF, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 8.0), and 1 mM PMSF. Cell lysates were precleared by the addition of protein G sepharose beads (Millipore, Billerica, MA, USA) and immunoprecipitated with protein G beads precoupled with an anti-Rab43 antibody (1:1,000, ab58030; Abcam, Cambridge, MA, USA). Immunoprecipitates or whole-cell lysates were fractionated by SDS-PAGE and transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). After blocking, the membranes were incubated with one of the following antibodies: anti-DLK1 (1:1,000, sc25437), anti-SCG2 (1:1,000, sc50290), anti-p-Ser (1:500, sc81514; Santa Cruz Biotechnology Inc.), anti-phosphorylated Akt (1:1000, #9271), anti-phosphorylated ERK1/2 (1:1,000, #9101), anti-ERK1/2 (1:1,000, #9107), anti-phosphorylated FOXO1 (1:1,1000, #9461), anti-FOXO1 (1:500, #2880; Cell Signaling Technology, Danvers, MA, USA), anti-PDX1 (1:1000, ab3503; Chemicon, Temecula, CA, USA), anti-synaptophysin (1:500, M0776; Dako Cytomation, Glostrup, Denmark), Lamin B1 (1:1000, 33–2000; Thermo Scientific, Rockford, IL, USA), or anti-beta-actin (1:5,000, A5441; Sigma Chemical Co.). Proteins were visualized using an enhanced chemiluminescence kit in accordance with the manufacturer’s recommendations.