Anti hur

RNA-immunoprecipitation assay was performed with Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore). The exponential cells were washed with ice-cold PBS and collected by centrifugation into complete RIP lysis buffer on ice for 5 min and followed by immediate freezing at −80 °C. The primary antibodies (anti-HuR, #12582, Cell Signaling Technology) were pre-bound to protein A/G magnetic beads on rotator at room temperature for 30 min, and then incubated with cell lysates overnight at 4 °C. After rigorous wash, the immunoprecipitated complexes were digested with proteinase K at 55 °C for 30 min. RNA species were recovered using TRIzol reagent and cDNA was prepared as previously described. Enriched HuR and GAPDH transcripts were analyzed by real-time PCR.

HK‐2 cells (Human, kidney proximal tubular 2) were obtained from ATCC (ATCC^® CRL‐2190™). The primary antibodies used in this study include anti‐PARKIN (Cat no: #2132, 1:500; Cell Signaling Technology), anti‐p‐PARKIN (Cat no: orb312554, 1:250, Biorbyt), anti‐PINK1 (Cat no: #6946, 1:500; Cell Signaling Technology), anti‐BNIP3L (Cat no: GTX111876, 1:1000, GeneTex), anti‐BNIP3 (Cat no: GTX10433, 1:1000, GeneTex), anti‐HuR (Cat no: #12582, 1:1000, Cell Signaling Technology), anti‐TOMM20 (Cat no: ab56783, 1:200, Abcam), anti‐LC3 (Cat no: NB100‐2220, 1:200, Novus Biologicals), anti‐COXIV (Cat no: 4850, 1:1000, Cell Signaling Technology), anti‐LAMP2 (Cat no: 49067, 1:200, Cell Signaling Technology), and anti‐β‐ACTIN (Cat no: A1978, 1:1000, Sigma‐Aldrich). Secondary antibodies‐rabbit/mouse IgG HRP, Alexa Fluor 546 anti‐mouse IgG (H + L), Alexa Fluor 488 anti‐goat IgG (H + L), Alexa Fluor 647 anti‐rabbit IgG (H + L) were purchased from Invitrogen™. 2′,7′‐dichlorofluorescin‐diacetate (DCFH2‐DA), JC‐1 and DAPI (4′,6‐diamidino‐2‐phenylindole) mount were purchased from Sigma Aldrich. Magna RIP™ RNA‐Binding Protein Immunoprecipitation Kit was procured from Sigma‐Aldrich. The SuperSignal West Femto Maximum Sensitivity Substrate detection reagents were purchased from Thermo Scientific Inc.

The protein expression levels of p53, FOXO3a, HuR, RPS19BP1, DBC1 in brain tissues were analyzed using western blotting analysis. The total protein of brain tissues was extracted using RIPA Lysis Buffer (Beyotime, China) according to the manufacturer’s instructions. Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime, China). 5 mg of proteins were separated using 12% SDS-polyacrylamide gels and transferred onto a PVDF membrane (0.45 μm, Millipore, USA). The blots were blocked with 5% non-fat milk in TBS, then incubated overnight at 4°C with the appropriate dilution of primary antibodies: anti-p53 (SC-6243, Santa Cruz Biotechnology), anti-FOXO3a (#12829, Cell Signaling Technology), anti-HuR (#12582, Cell Signaling Technology), anti-AROS (Ab201091, abcam), anti-DBC1 (#5857, Cell Signaling Technology), anti-β-action (AC004, ABclonal Technology). After washing the membranes to remove excess primary antibody, the membranes were incubated for 1 h at room temperature with the appropriate secondary antibodies at a dilution of 1:2000-1:5000 (AC004 or AS003, ABclonal Tehcnology). The membranes were washed 3 times and then visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific). β-action was used as an internal control.