Tris 2 carboxyethyl phosphine hydrochloride tcep

Agarose beads were washed twice in 1 mM Tris-HCl prior to elution of bound proteins. Samples were prepared for mass spectrometric analysis as previously described (31 (link)). Briefly, proteins were eluted from GFP-Trap beads by the addition of 20% (vol/vol) trifluoroethanol–formic acid (0.1%, pH 2.4) and were incubated at 50°C for 5 min. The eluate was reduced with 5 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Thermo Fisher Scientific) and neutralised with TEAB (tetraethylammonium bromide). Samples were digested with trypsin (Sigma) overnight at 37°C.
Samples were analyzed by electrospray ionization (ESI) LC-MS/MS on an Orbitrap Elite (purified Maurer’s clefts) or a Q Exactive (co-IP) mass spectrometer. Mass spectra (ProteoWizard) were searched against a custom database containing the Plasmodium falciparum 3D7 and UniProt human proteomes. Searches were performed on MASCOT (Matrix Science), with the following parameters: precursor ion mass tolerance of 10 ppm, fragment ion mass tolerance of 0.2 Da, trypsin as the cleavage enzyme, three allowed missed cleavages, and allowance for oxidation. The network map of protein interactions was created using NAViGaTOR 2.3 software, and the networks were redrawn in Adobe Illustrator.

The following primary antibodies are used: Mouse anti-Actin (Millipore, MAB 1510), Mouse anti-Myc 9E10 (Covance, MMs-150R), Mouse anti-Ubiquitin (Santa Cruz, sc-8017), Mouse anti-GFP (Roche, 11814460001), Rabbit anti-ZDHHC6 (Sigma, SAB1304457), Mouse anti-Transferrin Receptor (Thermo Scientific, 136800), Mouse anti-Flag M2 (Sigma, F3165), Rabbit anti-ANTXR1 (Sigma, SAB2501028), Rabbit anti-TRAPα (Abcam, ab133238), Rabbit anti-Flotillin1 were produced in our laboratory, Mouse anti-CLIMP63 (Enzo, ALX-804–604), Mouse anti-Calnexin (MAB3126), Rabbit anti-GP78 AMFR (Abnova, PAB1684), Rabbit anti-IP3R (Cell signaling, 85685). The following beads were used for immunoprecipitation: Protein G Sepharose 4 Fast flow (GE Healthcare, 17-0618-01), anti-Myc affinity gel (Thermo Scientific, 20169), anti-Flag affinity gel EZview M2 (Sigma, F2426). Drugs were used as follows: Bafilomycin A1 at 100 nM (Sigma, B1793), MG132 at 10 µM (Sigma, C2211), Hydroxylamine at 0.5 M (Sigma, 55459), mPEG-5k at 20 mM (Sigma, 63187), N-ethylmaleimide NEM at 20 mM (Thermo Scientific, 23030), Tris-2- carboxyethyl-phosphine hydrochloride TCEP at 10 mM (Thermo Scientific, 23225), Methyl methanethiosulfonate MMTS at 1.5% (Sigma, 208795), Puromycin at 3 µg/ml (Sigma, P9620).

Protein extracts for RPPA analysis were prepared as previously described [30 (link)]. Briefly, 6, 16, 24 and 36 h after treatment, cells were collected, washed twice in PBS and lysed in a home-made buffer containing T-PER reagent (Thermo Fisher Scientific, Waltham, MA, USA), 300 mM NaCl, protease and phosphatase inhibitors cocktails (Merck-Millipore, Burlington, MA, USA). Total protein concentration was measured using the Bradford reagent method (Bio-Rad Laboratories, Hercules, CA, USA). RPPA-ready protein extracts were prepared by diluting lysates in extraction buffer containing 47.5% T-PER, 50% 2X Sodium dodecyl sulfate (SDS) (Thermo Fisher Scientific) and 2.5% Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (Thermo Fisher Scientific) to a final concentration of 1 mg/mL in a volume of 40 mL. A further denaturation step of 5 min boiling was performed prior to freezing at − 80 °C. RPPA analysis was performed on a per service basis by the MD Anderson Cancer Center RPPA Core Facility, following their standard operating procedures [https://www.mdanderson.org/research/research-resources/core-facilities/functional-proteomics-rppa-core/education-and-references.html]. The list of antibodies utilized for RPPA analysis is available in Supplementary Table 2.

Anhydrous dimethyl sulfoxyde (DMSO, 99.9%), EDTA disodium salt, N-hydroxysuccinimide (NHS), Dicyclohexylcarbodiimide (DCC) and Dithiothreitol (DTT) were purchased from Sigma Aldrich (Saint-Quentin-Fallavier, France). α-Maleinimidohexanoic-ω-NHS PEG, Mw 5000 Da, (NHS-PEG-Mal) was obtained from Rapp Polymere (Tübingen, Germany). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and Coomassie Plus assay kit were purchased from Thermo Scientific (Fisher Scientific, Illkirch, France) and cyanine-5,5-NHS was obtained from Lumiprobe (Hannover, Germany). All other reagents were of analytical grade. In all the experiments, water was previously deionized (18 MΩ cm). Dialysis tubing (cellulose ester, molecular weight cut off 300 and 1000 kDa) was obtained from Spectrum Labs (France).