Fig. 4

STAU2 is hyperphosphorylated during the M-phase. a shRNA control (shCtrl) or against STAU2 (shSTAU2) were cloned in a retrovirus vector to infect hTert-RPE1 and HeLa cells. Cells were synchronized in prometaphase with nocodazole and mitotic cells were enriched by gentle shake off (Ndz + S.off). Protein extracts from asynchronous (−) and synchronized cells (+) were analyzed by western blotting to detect STAU2 migration and cell cycle markers (MPM2 and cyclins). β-actin was used as loading control. b Schematic representation of STAU2 expression vectors, STAU2⁵²-FLAG₃ and STAU2⁵⁹-FLAG₃. Dark gray boxes, double-stranded RNA-binding domains (dsRBD); light gray boxes, tubulin-binding domain (TBD); white boxes, FLAG₃. c hTert-RPE1 cells infected with viruses expressing the empty pMSCV vector (pMSCV), STAU2⁵²-FLAG₃, STAU2⁵⁹-FLAG₃ or both were synchronized (+) by nocodazole and shake off (Ndz + S.off). Migration of STAU2 proteins was detected by SDS-PAGE and western blotting. Both endogenous and overexpressed STAU2⁵⁹-FLAG₃ were analyzed with anti-STAU2 antibody, while anti-FLAG antibody was used to specifically recognize FLAG₃-tagged STAU2 isoforms. Mitotic marker accumulation was assessed with anti-MPM2 and anti-cyclin B1 antibodies. Loading was normalized with β-actin antibody. d Asynchronous (−) and nocodazole-treated (Ndz)(+) hTert-RPE1 cells were lysed and protein extracts were subjected to separation on phospho-columns. Input from total extracts (I), flow through (F) and phospho-eluates (P) were analyzed by western blotting using anti-STAU2. Anti-RSK1, anti-nucleolin and anti-β-actin antibodies were used as controls for phosphorylated and unphosphorylated proteins, respectively. e Protein extracts from nocodazole-treated (+) hTert-RPE1 and HeLa were incubated in vitro with either water (H2O), Lambda Phosphatase (λPP), inactivated Lambda Phosphatase (λPPin), Calf Intestinal Alkaline Phosphatase (CIP) or inactivated Calf Intestinal Alkaline Phosphatase (CIPin). STAU2 phosphorylation status was analyzed by SDS-PAGE and western blotting. Untreated cells (Mock) were used as control for dephosphorylated STAU2. All western blots are representatives of three independently performed experiments that showed similar profiles