Lamtor2

Whole cell extracts of BMDM, postnuclear supernatant, and LE/LYS samples were separated by SDS/PAGE, blotted and probed with the respective antibodies: LAMTOR2 from Cell Signaling (Frankfurt am Main, Germany), LAMP1 and EEA1 from BD Biosciences, tubulin from Sigma (Vienna, Austria), BiP from Gert Kreibich (New York University School of Medicine), phospho‐p70S6K (pT389), phospho‐S6 (S240/244), and S6 from Cell Signaling, p70S6K and SOAT1 from Santa Cruz (Heidelberg, Germany), MGLL from Thermo Scientific, actin from Merck.

For phosphorylation analysis of AKT, mTOR, p70 S6 Kinase 1, S6 ribosomal protein and ERK1/2, cells were stimulated for 20 min with Flt3 ligand. In addition, where stated, cells were treated with Rapamycin (50 ng ml⁻¹, LC Laboratories) or AC220 (100 ng ml⁻¹, LC Laboratories) for 1 h, before stimulation. Cell lysates of BMDCs and splenic DCs were prepared2 (link) and separated on a SDS–polyacrylamide gel electrophoresis. Membranes were probed after blotting and blocking against primary antibodies (LAMTOR2, LAMTOR3 (ref. 10 (link)), pAKT (1:500, clone D9E), AKT (1:500–1,000), pp70S6K1 (1:1,000, clone 1A5), pERK1/2 (1:500, clone E10), ERK1/2 (1:1,000, clone 137F5), pS6 (1:1,000, clone D57.2.2E) from Cell Signaling; Actin (1:5,000, clone C4) from Millipore; LAMTOR1 (1:1,000), LAMTOR4 (1:1,000) from Atlas; LAMTOR5 (1:500, clone C16) from Santa Cruz) overnight at 4 °C in blocking buffer (3% BSA, 1 mM EDTA, pH8, 0.05% Tween 20, 6 mM Sodium Azide, pH 5.2). Membranes were washed with Tris -buffered saline (50mM Tris-HCl, pH 7.6; 150mM NaCl) with 0.05% Tween 20 and incubated with the secondary antibodies (anti-mouse-/anti-rabbit IgG peroxidase antibody, 1:5,000 (Sigma)). Detection was performed by chemiluminescence. Uncropped western blots are provided in Supplementary Figs 7 and 8.

We performed western blotting analysis as previously described (17 (link)). Briefly, 30 µg of detergent soluble S1 protein was loaded to measure the relative abundance of the protein target. Protein samples were loaded onto the Novex™ 4 to 20% Tris-Glycine Plus 20-well Midi Protein Gels (1.0 mm, Thermo Fisher Scientific, Invitrogen™, #WXP42020BOXA). For a given probing target, all groups of mice were loaded to the same gel including resolving, transferring, and exposure. The following primary antibodies were used: Anti-Liver Arginase (abcam, #ab124917), LAMTOR2 (Cell Signaling Technologies, #8145), LAMTOR3 (Cell Signaling Technologies, #8168), LAMTOR4 (Cell Signaling Technologies, #12284), RagA (Cell Signaling Technologies, #4357), and anti-β-actin antibody (Sigma-Aldrich, #A5441). Densitometric analysis was performed using AlphaEase software (Alpha Innoch, CA, US).