Iblot system for 6 min

Retinas were lysed in 50 mM Tris pH8.0, 150 mM NaCl, 1 % NP-40 buffer containing protease inhibitors. Protein samples were separated by SDS-PAGE and electroblotted onto nitrocellulose membranes (Whatman) using the iblot System for 6 min (Invitrogen). Non-specific binding sites were blocked by incubation of the membrane with 5% non-fat milk in TBS containing 0.1% Tween 20 (TBST) for 1 h. Proteins were detected using the primary antibodies diluted in blocking solution (4% Bovine Serum Albumin in TBST) (Supplementary Tables 1 and 2 for list of antibodies). Following washing in PBST, blots were incubated with the appropriate secondary antibodies conjugated to horse-radish peroxidase (Pierce) and chemiluminescence detection of Super Signal West Pico detection reagent (Pierce) by high resolution image capture using the ImageQuant LAS4000 camera system (GE Healthcare). Images were transferred to Fiji/ImageJ and mean pixel intensity of protein bands measured for quantification, with an equal area of blot assessed across all bands.

Cell pellets were lysed in 50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40 buffer containing protease inhibitors. Protein samples either from ESCs or liver extracts were separated by SDS-PAGE and electroblotted onto nitrocellulose membranes (Whatman) using iblot System for 6 min (Invitrogen). Non-specific binding sites were blocked by incubation of the membrane with 5% nonfat milk in PBS containing 0.1% Tween 20 (PBST). Proteins were detected using the following primary antibodies diluted in blocking solution: mouse monoclonal anti-SRSF1 (clone 96; 1:1000; Hanamura et al., 1998 (link)), mouse monoclonal anti-tubulin (Sigma #T8328), and mouse monoclonal anti-T7 (Novagen, #69522, 1:10,000). Following washing in PBST, blots were incubated with the appropriate secondary antibodies conjugated to horse-radish peroxidase (Pierce) and detected with Super Signal West Pico detection reagent (Pierce).