Syvn1

Cells were lysed on ice in CelLytic MT Cell Lysis Reagent (Sigma) supplemented with protease and phosphatase inhibitors. Protein concentration was determined with Pierce BCA Protein Assay Kit (Thermo Fisher). Protein lysates were resolved on SDS-PAGE gels and transferred to PVDF membranes [36 (link)]. The following antibodies were used for the western blot analysis: phospho-eIF2α (#9721, 1:1000), eIF2α (#9722, 1:1000), ATF4 (#11815, 1:1000), BIM (#2819, 1:1000), SYVN1 (#14773, 1:1000) from Cell Signaling Technology; phospho-PERK (Thr980) (MA5–15033, 1:1000, Thermo Fisher); SEL1L (S3699, 1:1000, Sigma); β-tubulin (10094–1-AP, 1:5000, Proteintech).

The human breast cancer cell lines, MDA-MB-231, MDA-MB-468 and MDA-MB-453 representing triple negative breast cancer of Caucasian origin with no AR expression (QNBC), African origin with low AR and Caucasian origin with high AR expression respectively obtained from ATCC (Manassas, VA) were cultured in the recommended media supplemented with 10% heat-inactivated standard fetal bovine serum (FBS, GIBCO) and 1% penicillin-streptomycin (10,000 U/ml, Life Technologies) at 37°C and 5% CO₂. Primary antibodies of MNK1, MNK2, eIF4E, p-eIF4E, SYVN1, Ubiquitin, β-actin, GAPDH and secondary HRP-conjugated anti-rabbit were obtained from Cell Signaling Technology, USA. The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with 1x protease inhibitors (Roche, Indianapolis, IN, USA), phosphatase inhibitors (Thermo Scientific, Waltham, MA, USA), 1 mmol/L EDTA and 1 mmol/L PMSF (Sigma) and immunoblotted as described earlier (24 (link), 25 (link)). Immunoprecipitation of MNK1 was performed as reported previously using MNK1 primary antibody (26 (link), 27 (link)). All fine chemicals were purchased from Sigma-Aldrich, St. Louis, MO. VNLG-152R and the deuterated analogs (D6, D7 and H6) were synthesized in house as described previously (19 (link)). The chemical structures of VNLG-152R and its deuterated analogs are presented in Figure 1.