Complete mini edta free protease inhibitor tablet

P7 cortices were dissected in ice cold 1X PBS supplemented with cOmplete Mini, EDTA-free protease inhibitor tablets (Roche, 04 693 159 001) and transferred to RIPA buffer for 20 minutes on ice. The tissue was homogenized by pushing through 25G and 27G needles sequentially, 3 times each. Cell homogenate was centrifuged at 14,000 g for 10 minutes at 4°C. The supernatant was removed, denatured at 100°C for 5 minutes in 5x sample buffer, run on an 8% SDS-PAGE at 70V for 2 hours, transferred to PVDF (Sigma, IPVH85R) at 150mA for 90 minutes, blocked for one hour in 1% non-fat dry milk-TBST, and immunoblotted overnight with 2ug of FEZF2 (Rabbit, IBL F441) primary antibody. The blot was developed with Li-Cor Donkey anti Rabbit secondary antibody (#926-68073) for one hour and images were processed using ImageStudioLite.

Cells were trypsinized and centrifuged, then re-suspended in a lysis buffer (150 mM NaCl, 2 mM EDTA and 50 mM Tris-HCl, pH 7.6) containing 0.1% NP-40, complete mini-EDTA free protease inhibitor tablets (Roche) and phosphatase inhibitors (Sigma-Aldrich). Laemmli solution (5X, Sigma-Aldrich) was then added to a final concentration of 1X and were heated for 10 minutes in a boiling water bath.
SDS-PAGE was carried out on a 4–12% acrylamide gel gradient before transferring proteins onto a polyvinylidene fluoride membrane (GE Healthcare). Western blotting was done using rabbit monoclonal anti-5-LO (1:500 in TBS-Tween; Cell Signaling Technology), rabbit monoclonal anti-hemagglutinin (1:2000 in TBS-Tween; Cell Signaling Technology), rabbit anti-phospho-S523 5-LO (1:500 in TBS-Tween; Cell Signalling Technology), rabbit anti-phospho-S271 5-LO (1:500 in TBS-Tween; Cell Signalling Technology), and a horseradish-conjugated mouse anti-rabbit IgG (Jackson Immunoresearch). Membranes were then developed using ECL prime western blotting detection reagent (GE Healthcare) and detection was performed using an Alpha Innotech Fluorchem imager.

Digoxygenin-O-methylcarbonyl-ε-aminocaproic acid N-hydroxysuccinimide (DIG) ester (#11333054001), anti-DIG-POD Fab fragments (#11207733910) and cOmplete Mini EDTA-free protease inhibitor tablets (#1697498) were from Roche; Coomassie Protein Assay reagent from Thermo Scientific, polyethylenimine (PEI) from Polysciences Inc; Protein G-Sepharose, glutathione-Sepharose and enhanced chemiluminescence Western blotting kit (RPN2106) were from Amersham Bioscience; Precast NuPAGE® polyacrylamide Bis-Tris gels, Lipofectamine® 2000 transfection reagent, hygromycin, blasticidin, zeocin, puromycin, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (D1306) and Alexa Fluor® Phalloidin (A12381) were from Invitrogen; sequencing-grade trypsin from Promega; and microcystin-LR was purchased from Professor Linda Lawton (Robert Gordon University, Aberdeen, U.K.).

negGFP-ANKS3-SAM fusion transformed into ARI814 cells [45 (link)] was expressed as described previously [22 (link)]. Harvested cells were lysed as described previously, except that 5 cOmplete mini, EDTA-free protease inhibitor tablets (Roche) were included in the lysis buffer and five 1-min. rounds of sonication were used [22 (link)]. Following centrifugation at 13,200 g for 20 minutes, lysate supernatant was supplemented with 10 mM imidazole and rocked with 2 mL of Ni-NTA Superflow agarose (Qiagen) for 1 hour at 4°C. The resin was washed with lysis buffer (20 mM Tris pH 7.5, 1 M NaCl, 1 mM TCEP) containing 20 mM imidazole, and eluted with lysis buffer containing 75 mM imidazole. Eluted protein was dialyzed into 20 mM Tris pH 7.5, 0.15 M NaCl, 1 mM TCEP and loaded onto a 5 mL HiTrap Q HP column (GE) 4°C. Protein was eluted using a shallow gradient of NaCl (0.25-0.5 M) in 20 mM Tris pH 7.5, 1 mM TCEP. Purified protein was dialyzed into 20 mM Tris pH 7.5, 0.3 M NaCl, 1 mM TCEP and concentrated using an Amicon Ultra (10 kDa MWC0) centrifugal filter unit (Millipore) to a final concentration of 4.3 mg/mL.

Hippocampi from E16 embryos were dissected individually into Eppendorf tubes and flash frozen. The adult hippocampus from a male mouse was dissected and flash frozen. Adult cortex from a male mouse was flash frozen. Samples were stored at −80°C until lysate preparation.
To prepare lysate, frozen tissue was transferred to a 2 mL Dounce homogenizer filled with ribosome-profiling lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, 100 mM KCl, 1% Triton-X 100, 2 mM DTT, 100 µg/mL cycloheximide, 500 U/mL RNasein Plus [Promega], and cOmplete Mini EDTA-free Protease Inhibitor Tablets [Roche, 1 tablet/10 mL]) (Subtelny et al. 2014 (link)). Samples were then homogenized with 10 strokes of pestle A followed by 10 strokes of pestle B, taking care not to introduce bubbles into the buffer. Following homogenization, the sample was transferred to two Eppendorf tubes, centrifuged at 1300g for 10 min, and the supernatant was aliquoted and flash frozen at −80°C for use in ribosome profiling, RNA sequencing, and tail sequencing. For RNA and tail sequencing, RNA was extracted by adding five volumes of TRIzol (Thermo Fisher) to the frozen lysate, allowing it to thaw to room temperature, and continuing the preparation according to the manufacturer's instructions.