Anti gfp 2956

Fifty microlitre of GFP-Trap A agarose beads (Chromotek) was washed in 500 μl buffer (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% SDS and 0.5% NP-40) containing a protease inhibitor cocktail. Five milligram of relevant GFP-tagged protein lysate was mixed with prewashed GFP beads and incubated overnight at 4 °C. Beads were subsequently washed on ice three times with washing buffer. For elution, the washed beads are resuspended in 100 μl of 2 × LDS sample buffer (Thermo Scientific) and boiled at 95 °C for 5 min. The eluate was loaded onto SDS page gel followed by western blot analysis using standard protocols. For western blot analysis, the following primary antibodies were used: anti-GFP (sc-9996, Santa Cruz), anti-GFP (2956, Cell Signaling), streptavidin-HRP (3999, Cell Signaling), anti-Gapdh (ab8245, Abcam) and anti-actin (ab8224, Abcam). Anti-H3K56 acetylation (39281; Activemotif), anti-K-acetylation (9441, Cell Signaling), anti-K-succinylation (PTM-401, PTM Biolabs), anti-Porin (16G9E6BC4, Life technologies, Carlsbad, CA, USA) and HRP-coupled anti-mouse or anti-rabbit secondary antibodies were from Jackson Immunoresearch. All western blots presented in the main text have been included as uncropped scans in Supplementary Fig. 6.

Cells were lysed in Laemmli buffer, proteins were separated on SDS-PAGE, and transferred to polyvinylidene fluoride membranes (Bio-Rad). Primary antibodies used were anti-FLAG (2368; NEB; 1:1000 dilution), anti-HA (3724; NEB; 1:1000 dilution), anti-mTOR (7C10; Cell Signaling; 1:1000 dilution), anti-DEPTOR (D9F5; Cell Signaling; 1:1000 dilution), anti-S6 (54D2; Cell Signaling; 1:1000 dilution), anti-phospho-S6^S240/244 (2215; Cell Signaling; 1:1000 dilution), anti-pan-AKT (C67E7; Cell Signaling; 1:1000), antiphospho-AKT^S473 (D9E; Cell Signaling; 1:1000 dilution), anti-ACTIN (D6A8; Cell Signaling; 1:2000 dilution) and anti-GAPDH (D16H11; Cell Signaling; 1:2000 dilution), anti-4G10 (05-321; Millipore–Sigma; 1:1000 dilution), anti-GFP (2956; Cell Signaling; 1:1000 dilution), antiubiquitin P4D1 (sc-8017; Santa Cruz; 1:1000 dilution), anti-EPHB2 D2X2I (83029; Cell Signaling; 1:1000 dilution), anti-EPHB2 (AF467; R&D Systems; 1:200 dilution), and anti-SYK D3ZE1 (13198; Cell Signaling; 1:500 dilution).