Critical roles of Astrin in the mitosis of immature rat Sertoli cells

Loss of Wwox Causes Defective Development of Cerebral Cortex with Hypomyelination in a Rat Model of Lethal Dwarfism with Epilepsy

“…Following three washes with PBS, tissue sections were blocked with 10% donkey serum (Sigma-Aldrich) in PBS for 30 min and incubated overnight at 4 °C with primary antibodies. After washing with PBS, the sections were incubated with appropriate secondary antibodies [55,56] for 1 h at room temperature. The resultant sections were mounted in ProLong Gold Antifade Reagent containing DAPI (Life Technologies, Carlsbad, CA, USA) for counterstaining.…”

“…Proteins in the extracts were separated by SDS-PAGE and transferred to PVDF membranes (Hybond-LFP, GE Healthcare Life Science, Pittsburgh, PA, USA) using semi-dry blotting system (ATTO, Tokyo, Japan) at 25 V for 15 min as described [13,55]. After blocking with 1% non-fat milk in PBS containing 0.1% Tween 20 for 1 h, the membranes were incubated overnight at 4 °C with appropriately diluted primary antibody, as above, followed by incubation with appropriate secondary antibodies and visualization with an Odyssey fluorescent imaging system (version 1.2; LI-COR Biotechnology, Lincoln, NE, USA) [55]. Densitometric analyses on western blot were performed by software Image J as previously described [55].…”

Loss of Wwox Perturbs Neuronal Migration and Impairs Early Cortical Development

“…Before removing the brain, whole body was perfused with phosphate-buffered saline (PBS) containing 10 unit/mL heparin followed by 4% buffered paraformaldehyde. After 48 h fixation with paraformaldehyde, isolated brains were embedded in O.T.C compound according to general protocol as previously described (Tochigi et al, 2017). Twenty-µm-thick sequential sagittal cryosections were prepared from 1 to 2 mm lateral region to the midline.…”

“…Anti-Satb2 (1:500, clone SATBA4B10, Abcam plc, Cambridge, United Kingdom) and anti-Tbr1 (1:5000, ab31940, Abcam plc) were used to detect layer specific-neuron subtypes (Hevner et al, 2001;Britanova et al, 2005). The positive signals of BrdU were visualized by denaturing with HCl treatment and following immunohistochemical procedure previously described (Tochigi et al, 2017). All histological images were taken on a Biozero BZ-X710 fluorescence microscope using software BZ-X analyzer Ver.…”

Proteins and Proteoforms: New Separation Challenges

“…Polyclonal antibodies that target PTMs are likely to be of more limited value in the selection of proteoform families for this reason. Monoclonal antibodies that target PTMs will in contrast be very useful as has been found with antihistone H3 (dimethyl-K4), 57 antihistone H3 (phospho-S10), 58 and antihistone H3 (acetyl-K4) 58 antibodies.…”