32p orthophosphate

[γ-³²P]ATP (3,000 Ci/mmol) and [³²P]orthophosphate were purchased from PerkinElmer Life Sciences (Turku, Finland). HUVECs were purchased from the Medical Center for Cells, Central South University (Changsha, China). Pertussis toxin (PTX), NS398, and 4′-6-diamidino-2-phenylindole were obtained from Sigma (St. Louis, USA). GF109203X, PD98059, JTE013, and VPC23019 were from Cayman (Michigan, USA). Competitive enzyme immunoassay kit for 6-keto PGF1α, nuclear and cytoplasmic protein extraction kit, and chromatin immunoprecipitation (ChIP) assay kit were obtained from Beyotime Institute of Biotechnology (Haimen, China). Mouse GAPDH was from Boster (Wuhan, China). Rabbit anti-COX-2 antibodies were from Epitomics (Burlingame, USA). Rabbit polyclonal anti-SphK-2 and anti-SphK1 antibodies were from Abcam (Cambridge, USA). P-CREB, ERK1/2, P-ERK1/2, P-PKCα, and PKCα antibodies were from Cell Signaling (Boston, USA). HDL was purchased from Millipore Corporation (Billerica, USA). Lipofectamine 2000 was from Invitrogen (Carlsbad, USA). The small interfering RNA (siRNA) for SphK-2 was obtained from Ruibo Co., Ltd. (Guangzhou, China). RealSuper Mixture (with ROX) was obtained from Cowin Biotech Co., Ltd. (Beijing, China).

2-Aminoethoxydiphenylborate (2-APB), thapsigargin, Latrunculin A, U-73122 were purchased from Calbiochem. m-3M3FBS, and neomycin were from Sigma-Aldrich. The PBX calcium assay kit, the antibodies against CD3ε (clone 145- 2C11), the F23.1 anti-TCR Vβ 8 1–3 antibody were supplied by Becton Dickinson. The FITC-conjugated anti-PI(4,5)P2 and anti-PI4P mAbs were from Echelon Biosciences. Alexa Fluor^TM 488-conjugated Dnase1 and Alexa Fluor^TM-conjugated phalloidin were supplied by Life technologies (Molecular probes). The anti-Vβ8 TCR (F23.2), anti-Cβ (H57-597), anti-CD3ε (145-2C11), anti-CD4 (GK1.5), anti-CD45 (H193.16.3), anti-Thy-1 (H194.92) (10 mg/mL, final) were purified from cell culture supernatants of the hybridomas in the lab; according to standard protocols. The antibodies purchased for immunoblotting were as follows: mouse anti-phosphotyrosine (4G10; Merck Millipore); anti-Zap-70 (clone 29/Zap70, BD Biosciences); anti-phospho-Zap70 (Tyr319) (Cell Signaling). H146-968 (anti-CD3ζ) mAb was produced in the lab. All other chemical reagents were from Sigma–Aldrich. [³²P]orthophosphate was from PerkinElmer.

All chemicals were reagent grade. Phospholipid standards were purchased from Avanti Polar Lipids. Neutral lipid standards were purchased from Nu-Chek Prep. Deuterium-labeled glycerol (1,1,2,3,3-D5, 99%) was purchased from Cambridge Isotope Laboratories, Inc. [³²P]-orthophosphate was purchased from PerkinElmer, and scintillation-counting supplies were purchased from National Diagnostics. Silica gel loaded SG81 chromatography paper was purchased from Whatman, Inc, and HPTLC plates from Merck. Growth medium supplies were purchased from Difco Laboratories.

Relative quantities of (p)ppGpp were measured by TLC of radiolabeled nucleotides, as previously described (30 (link)). B. burgdorferi 297 wild-type, the isogenic ΔdksA mutant, and ΔdksA pDksA mutant strains were cultured to 1 × 10⁸ spirochetes ml⁻¹ in BSK II medium containing 20 µCi/ml [³²P]orthophosphate (PerkinElmer, Waltham, MA) in 500 µl, pelleted by centrifugation at 9,000 × g for 7 min, and resuspended in RPMI 1640 medium. Cultures were collected by centrifugation at 20,800 × g for 5 min at 4°C, cells were washed once with Dulbecco’s phosphate-buffered saline (dPBS), and the cell pellet was lysed with 6.5 M formic acid (Thermo Fisher Scientific, Grand Island, NY). Cell debris was removed by centrifugation at 20,800 × g for 5 min at 4°C. The nucleotides were separated by TLC on polyethylenimine cellulose plates (EMD Millipore, Burlington, MA) in 1.5 M KH₂PO₄ (pH 3.4) buffer. After drying the TLC plates, radioactivity was detected by a 48- to 72-h exposure to an intensifying screen, and screens were imaged by a FLA-3000G phosphorimager (Fujifilm Life Sciences, Stamford, CT). Values are expressed as the ratio (p)ppGpp/[(p)ppGpp + GTP] from the densitometry measurements from three independent experiments. The mean values from three independent experiments were analyzed using one-way ANOVA and Tukey’s post hoc test to determine if differences were statistically significant.

In vivo metabolic labeling was performed as previously described (Burger and Eick, 2016 (link)). HEK293 cells were depleted from the endogenous phosphate pool by preculture in OptiMEM (Gibco) for 2 h. 15 µCi/ml [³²P]orthophosphate (3,000 Ci/mM; PerkinElmer) and DNA-damaging agents were added simultaneously and incubated for an additional 2 h. Dicer was immunoprecipitated from whole cell lysates. De novo phosphorylation was analyzed by autoradiography after calf intestine phosphatase (CIP; Invitrogen) treatment using 1 U for 1 h at 37°C. Upon separation by SDS-PAGE, signals were visualized by autoradiography and quantified using a Phosphorimager (Fujifilm) and AIDA software. CIP was visualized using a silver staining kit (Invitrogen), according to the manufacturer’s protocol.