Phospho ndrg1 thr 346

The following antibodies were used in this study: 4EBP1 (catalog #9644, 1:4000 dilution), Phospho-4EBP1-Thr37/46 (#2855,1:1000), Phospho-4EBP1 -Ser65 (#9451,1:1000), p70S6 Kinase (#2708,1:1000), Phospho-p70S6 Kinase-Thr389 (#9234,1:1000), Phospho-S6-Ser240/244(#5364,1:2000), AKT(#4691,1:2000), Phospho-AKT-Ser473 (#4060,1:1000), EIF4E (#9742,1: 2000), EIF4G (#2498,1:1000), NDRG1(#5196,1:2000), Phospho-NDRG1-Thr 346(#5482,1:2000), Phospho-Tuberin/TSC2-Ser939 (#3615,1:1000), SGK3 (#85731:1000), Phospho-SGK3-Thr320 (#5642,1:500), Survivin (#2808, 1:1000) and Rictor (#2114,1:1000), and were purchased from Cell Signaling Technology (Danvers, MA). In addition, γ-Tubulin (sc-7396,1:500) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Tuberin (ab32554,1:1000) and INPP4B (ab81269, 1:1000) were purchased from Abcam (Cambridge, UK).

BMDMs were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with a 1× protease/phosphatase inhibitor mixture (Thermo Fisher Scientific). Protein concentrations were estimated using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Equal amounts of protein were separated on 10% or 12% SDS/PAGE gels. The proteins were then transferred onto a polyvinylidene difluoride membrane and probed with the following antibodies: Lepr (ab5593, Abcam), phospho-Stat3 (9138, Cell Signaling), Stat3 (9139, Cell Signaling) Lamp-1 (sc-17768, Santa Cruz Biotechnology), phospho-Akt Ser473 (4060, Cell Signaling), phospho-Akt Thr-308 (2965, Cell Signaling), Akt (4691, Cell Signaling), phospho-Ndrg1 Thr-346 (3271, Cell Signaling), Rictor (2140, Cell Signaling), Phllp1 (07-1341, Merck Millipore), phospho-mTOR Ser-2448 (2971, Cell Signaling), phospho-s6K (9205, Cell Signaling) and Gapdh (2118S, Cell Signaling). Gapdh was used as a loading control. For phagosome samples, Ponceau staining was used as loading control (sc-301558, Santa Cruz Biotechnology). After incubation with secondary horseradish peroxidase-conjugated antibodies (R&D), the blots were developed using enhanced chemiluminescence reagent (GE Healthcare).