Anti flag agarose beads a 2220

Anti-HDAC6 (7612), mouse monoclonal anti-Myc-Tag (2276), rabbit polyclonal anti-Myc-Tag antibody (2278), anti-Cullin 3 (2759), anti-LC3-B (2775), EMT antibody sampler kit (9782), anti-Ac-α-Tubulin (5335) and anti-GST (2625) antibodies were purchased from Cell Signaling. Anti-TRIM24 (TIF1α, SC-271266), anti-Cullin 1 (SC-11384), anti-HA antibody (SC-805), anti-Tubulin (SC-73242) and anti-p27 (SC-527) antibodies were purchased from Santa Cruz. Anti-SPOP antibody (16750-1-AP) was purchased from Proteintech. Anti-GFP (8371-2) antibody was purchased from Clontech. Polyclonal anti-Flag antibody (F7425), monoclonal anti-Flag antibody (F-3165, clone M2), anti-vinculin antibody (V-4505), peroxidase-conjugated anti-mouse secondary antibody (A-4416), peroxidase-conjugated anti-rabbit secondary antibody (A-4914), anti-HA agarose beads (A-2095) and anti-Flag agarose beads (A-2220) were purchased from Sigma. All antibodies were used in 1:1000 dilutions in 5% non-fat milk for western blot.
MLN4924 was a kind gift from Dr. William Kaelin (Dana-Farber cancer institute). Bafilomycin A1 was kindly provided by Dr. Junying Yuan (Harvard Medical School). MG132 (BML-PI102-0005) was purchased from Enzo life science.

Cells were rinsed with ice-cold phosphate-buffered saline (PBS) and lysed with EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) or Triton buffer (40 mM HEPES pH 7.4, 150 mM NaCl, 2.5 mM MgCl₂, and 1% Triton) supplemented with protease inhibitor (Thermo Fisher, A32953) and phosphatase inhibitors (phosphatase inhibitor cocktail sets I and II, Calbiochem). The cell lysates were centrifuged at 13,200 r.p.m. for 10 min at 4 °C. The protein concentrations were measured using Nanodrop (Thermo Fisher) with Bio-Rad protein assay reagent. Equal amounts of whole cell lysates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with the indicated antibodies. For immunoprecipitation, the lysates (1000–3000 μg) were incubated with 50% slurry of agarose conjugated antibody for 3–5 h at 4 °C. The immunoprecipitates were washed four times with NETN buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.5% NP-40) or Triton buffer before being resolved by SDS-PAGE. Anti-FLAG agarose beads (A2220) and anti-HA agarose beads (A2095) were purchased from Sigma. Anti-AKT1 antibody conjugated to agarose (sc-5298 AC) and anti-PRMT5 antibody conjugated to agarose (sc-376937 AC) were purchased from Santa Cruz Biotechnology.

Mouse monoclonal anti-Myc-Tag (2276), rabbit monoclonal anti-Myc-Tag antibody (2278), anti-β-TRCP (4394), anti-Cyclin D1 (2978), anti-LRP6 (3395), rabbit polyclonal anti-Cullin 1 (4995) antibodies were purchased from Cell Signaling. Anti-HA antibody (SC-805), anti-Tubulin (SC-73242), anti-c-Myc (SC-40), anti-Axin-2 (SC-8570) and anti-p27 (SC-527) antibodies were purchased from Santa Cruz. Anti-ZNRF3 antibody (ab176449) was purchased from Abcam. Anti-GFP (8371-2) antibody was purchased from Clontech. Anti-β-catenin (AV100600), polyclonal anti-Flag antibody (F7425), monoclonal anti-Flag antibody (F-3165, clone M2), anti-Vinculin antibody (V-4505), peroxidase-conjugated anti-mouse secondary antibody (A-4416), peroxidase-conjugated anti-rabbit secondary antibody (A-4914), anti-HA agarose beads (A-2095) and anti-Flag agarose beads (A-2220) were purchased from Sigma. All antibodies were used in 1:1000 dilutions in 5% non-fat milk for Western blot. Proteasome inhibitor MG132 (S2619) and CKI inhibitor D4476 were obtained from Selleckchem. CHX (C4859) was purchased from Sigma; the neddylation inhibitor MLN4924 was a kind gift from Dr. William Kaelin (Dana-Farber cancer institute). λ-PPase (P0753S) was obtained from New England Biolabs.

Cells were rinsed with ice-cold phosphate-buffered saline (PBS) and lysed in EBC buffer (50 mM Tris–HCl pH 7.5, 120 mM NaCl and 0.5% NP-40) or Triton buffer (40 mM HEPES pH 7.4, 150 mM NaCl, 2.5 mM MgCl₂, 1 mM EDTA and 1% Triton X-100) supplemented with protease inhibitor (Thermo Fisher, A32953) and phosphatase inhibitors (phosphatase inhibitor cocktail Set I and II, Calbiochem). The cell lysates were centrifuged at 13,200 r.p.m. at 4 °C for 10 min. The protein concentration of lysates was determined using Nanodrop by Bio-Rad protein assay reagent. Equal amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For IP, 2000–5000 μg lysates were incubated with agarose conjugated antibodies for 3–5 h at 4 °C. Immunoprecipitants were washed three times with NETN buffer (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA and 0.5% NP-40) or Triton buffer before being resolved by SDS-PAGE. Anti-HA agarose beads (A2095) and anti-FLAG agarose beads (A2220) were purchased from Sigma-Aldrich. Anti-Myc agarose beads (658502) were purchased from BioLegend. Some blots were cut prior to hybridization with primary antibodies, but one full-length original, unprocessed blot for each antibody was provided in the Supplementary Materials.

Anti-β-TRCP1 (4394), anti-AKT (2920), anti-phospho-AKT (4060), anti-mTOR L27D4 (4517), anti-mTOR 7C10 (2983), anti-phospho-mTOR (5536), anti-S6K (2708P), anti-phospho-S6K (9234P), anti-DEPTOR (11816), anti-FLCN D14G9 (3697), and anti-GFP (2955) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-c-Myc 9E10 (sc-40), rabbit polyclonal anti-HA (sc-805), anti-p27 (sc-528), anti-SKP2 (sc-74477), anti-Actin (sc-47778), and anti-FLCN FL-342 (sc-25777) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal anti-Flag antibody (F-1804), peroxidase-conjugated anti-mouse secondary antibody (A-4416), peroxidase-conjugated anti-rabbit secondary antibody (A-4914), and anti-Flag agarose beads (A-2220) were purchased from Sigma-Aldrich. Monoclonal anti-HA antibody (MMS-101P) was purchased from Covance (Princeton, NJ, USA). Anti-FNIP1 antibody (ab134969) was purchased from Abcam (Cambridge, MA, USA). Anti-FNIP2 antibody (HPA042779) was purchased from Atlas Antibodies AB (Stockholm, Sweden).