Neg772002mc

Stable MEF cell lines overexpressing MARK2^WT or MARK2^T595A were plated onto 6-well plates (2 × 10⁵ cells per well) overnight. For puromycin labeling, cells were treated with 10 ug/ml puromycin in culture medium for 10 min and then washed 3 times with 1 X PBS and lysed with RIPA buffer as described above. The cell lysate was analyzed by immunoblotting against puromycin. For ³⁵S labeling, cells were incubated with methionine- and cysteine-free DMEM supplemented with 10% FBS (MilliporeSigma F0392) for 1 h. A total of 200 μCi of [³⁵S]-methionine and [³⁵S]-cysteine (PerkinElmer NEG772002MC, USA) was then added to each dish to metabolically label the cells for 1 h. After radiolabeling, the cells were washed 3 times with 1 X PBS and lysed with RIPA buffer as described above. A total of 20 ng of cell lysate was added into 4 ml of liquid scintillation cocktail (MP Biomedicals 01882475-CF, USA), and the radioactivity was detected using LS6500 Liquid Scintillation Counter (Beckman Coulter, USA).

After an overnight fast, mice were given an intraperitoneal injection of 200 µCi of [³⁵S] methionine/cysteine protein labeling mix (NEG772002MC, Perkin Elmer) combined with 1,000 mg/kg pluronic F127 poloxamer-407 (BASF, P2443, Sigma-Aldrich) to inhibit lipoprotein clearance from plasma as previously described^{58 (link)}. Triglyceride secretion was calculated using plasma collected at 2 h post-injection. Total plasma apoB100 and apoB48 secretion was determined by taking 2 µl of plasma from the 2 h time point and separating the proteins by SDS-PAGE gel electrophoresis followed by densitometric quantification using ImageJ. Plasma from Apobec1^−/− mice (which express only apo B100) was used as control to identify apoB100 and apoB48 bands^{59 (link),60 (link)}.

Organoids were replenished with medium 6 hr prior to analysis while in the optimal growth phase post-splitting. Technical triplicates were used from organoids from three different animals per experiments. ³⁵S-methionine (Perkin Elmer #NEG772002MC) was used at 30 µCi/ml for 30 min. Samples were lysed using the same buffer described for Western blotting. Protein was precipitated in 12.5% (wt/vol) trichloroacetic acid onto glass microfiber paper (Whatmann #1827-024) by use of a vacuum manifold. Precipitates were washed with 70% ethanol and acetone. Scintillation was read on a Wallac MicroBeta TriLux 1450 scintillation counter using Ecoscint scintillation fluid (SLS Ltd #LS271) from these microfiber papers. In parallel the total protein content was determined by BCA assay using unprecipitated sample. Protein synthesis rate was expressed as the scintillation normalised to the total protein content (CPM/µg/ml protein), which was then changed to a relative value compared to relevant controls for each experiment.