Tgx precast gel

Culture supernatants were subjected to electrophoresis on TGX Precast gels (BioRad), proteins were transferred to a PVDF membrane, which was blocked with PVDF Blocking Reagent (Toyobo Co. Ltd., Osaka, Japan), then incubated with a rabbit IgG anti-OPG antibody (GeneTex, Irvine, CA, USA). After thorough washing with 0.5% Tween-20 in PBS (PBS-T), the membrane was incubated with a horseradish peroxidase-conjugated anti-rabbit IgG antibody (R&D Systems, Inc., Minneapolis, MN, USA). Chemiluminescence was produced by using Luminata-Forte (EMD Millipore, Billerica, MA) and detected with LumiCube (Liponics, Tokyo, Japan).

Lipoplex-protein complexes
were resuspended in 40 μL of Laemmli Loading buffer 1×
and boiled for 10 min at 100 °C. Each sample was loaded on a
gradient polyacrylamide gel stain free (4–20 % TGX precast
gels, BioRad) and run at 100 V for about 150 min. Finally, gel images
were acquired with a ChemiDoc gel imaging system (Bio-Rad, CA) and
processed by means of custom MatLab scripts (MathWorks, Natick, MA),
as previously reported.^{55 (link)}

Protein was extracted from cells and posterior eyecup without conjunctiva, sclera, and muscle tissue with RIPA buffer containing protease inhibitor. The concentration of soluble protein was measured using BSA as standard (SpectraMax iD5, Molecular Devices, Sunnyvale, CA, USA). Equal amounts of protein (30 μg) were resolved on TGX-precast gels (Bio-Rad Laboratories Inc., Hercules, CA, USA) and transferred to PVDF blotting membranes (Millipore, Billerica, MA, USA). Membranes were probed with respective primary antibodies overnight at 4 °C (The antibodies and dilutions used are listed in Table 2). After incubation with the appropriate secondary antibodies (Vector Laboratories, Burlingame, CA, USA), protein bands were visualized by a chemiluminescence (ECL) detection system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Equal protein loading was confirmed with GAPDH. Protein band intensity was measured by Image J software (Version 1.51 23 April 2018 and URL accessed on 12 April 2018) (https://imagej.nih.gov/ij/). The quantification indicates the relative amounts as a ratio of each protein band to the appropriate loading control and expressed arbitrary units [35 (link)].

Polyacrylamide gel electrophoresis was used to resolve proteins from cellular lysates harvested in RIPA buffer. Protein determinations (BCA) were performed to ensure equal loading of protein on a per-sample basis. QKI-5, -6 and -7 were detected using primary mouse monoclonal antibodies that target pan-QKI (1:1,000, N73/168; UC Davis/NIH NeuroMab Facility), QKI-5 (1:1,000, N195A/16; UC Davis/NIH NeuroMab Facility), QKI-6 (1:1,000, N182/17; UC Davis/NIH NeuroMab Facility) or QKI-7 (1:1,000, N183/15.1; UC Davis/NIH NeuroMab Facility), or polyclonal antibodies targeting QKI-5 (1:1,000, AB9904; Millipore, Amsterdam, The Netherlands), QKI-6 (1:2,000, AB9906; Millipore) and QKI-7 (1:2,000, AB9908; Millipore). For loading references, rabbit polyclonal antibodies were used to detect β-actin or Histone H3 (both 1:4,000; Abcam, Cambridge, UK). All gels were run and blotted with Bio-Rad TGX pre-cast gels and blotted on nitrocellulose 0.2 μm using the Bio-Rad TurboBlot system (Bio-Rad Laboratories). Full blots are shown in Supplementary Fig. 7.