En3hance solution

HEK293 cells were seeded on 35 mm dishes and allowed to grow to a confluency of 80% at 37°C, 5% CO_2. Cells were transfected with pcDNAflux3_ApoER2_HA. After 24 h cells were shifted to Dulbecco’s Modified Eagle’s Medium without L-methionine and L-cysteine supplemented with 10% fetal calf serum for 30 min. Then, cells were labeled with 200 μCi/mL EasyTag™ EXPRESS³⁵S Protein Labeling Mix (PerkinElmer) for 30 min (pulse period) followed by the chase period for the indicated time intervals in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. After the chase period, the cells were washed twice with 2 ml of ice-cold PBS and then lysed by addition of 0.3 ml of NP-40 lysis buffer supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail and 1 mM EDTA. ApoER2_HA was immunoprecipitated from the lysates using anti-HA plus Protein G Sepharose (Invitrogen, Carlsbad, CA, USA) over night at 4°C. The immunoprecipitates were dissolved by heating at 95°C for 10 min in 4× SDS Protein Sample Buffer and then subjected to electrophoresis on 8% or 5% SDS-PAGE followed by either fixation, treatment with EN3HANCE solution (PerkinElmer, Waltham, MA, USA), and drying for autoradiography, or processed for western blotting using Ab20 as described above.

AdoMet cross-linking was performed as described^{8 (link)}. An aliquot of 2 µg of purified enzyme with 0.5 µCi of [methyl-³H]AdoMet (17.9 Ci/mmol, PerkinElmer) in 20 µl of 50 mM BisTris propane (pH 8.5), 1 mM EDTA, and 0.5 mM DTT, were transferred to a 96-well plate on ice and placed 8 cm from an inverted UV transilluminator (Compact UV Lamp, UVGL-25 (Analytik Jena US)) using UV-C (254 nm wavelength) for 1 h. The protein was then separated by SDS-PAGE, stained with Coomassie, and subjected to fluorography. The gels were incubated in water for 30 min, then incubated in 5× volume of EN3HANCE solution (Perkin Elmer) for 1 h. Gels were washed in 1% glycerol for 1 h and dried. An X-ray film (Amersham Hyperfilm MP (Cytiva)) was exposed to the dried gel for 48 h at −80 °C, followed by development of the X-ray film.

Parasites were washed three times in phosphate buffered saline (PBS 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ and 1.8 mM KH₂PO₄) and resuspended in methionine-free RTH/FCS supplemented with or without 50 μg ml⁻¹ cycloheximide for 30 min prior to the addition of 10 μCi ml⁻¹ L-[³⁵S]-methionine (Perkin Elmer). Lysates were made in Laemmli buffer and separated by SDS-PAGE as described above. The gel was treated with En3Hance solution (Perkin Elmer) as per manufacturer’s protocol, dried and exposed to BioMax MS film (Kodak) using a TransScreen LE (Kodak).