Gradient precast protein gel

Protein from approximately 500 human pseudo islets was extracted by sonication in RIPA buffer (Thermo Scientific) supplemented with a Protease inhibitor cocktail (Thermo Scientific). Samples were denatured and resolved on a 4–15% gradient precast protein gel (Biorad). Proteins were transferred onto a PVDF membrane (Biorad) and blocked with 5% non-fat dry milk (Thermo Scientific). Membranes were probed for a series of proteins with the following antibodies: rabbit anti-BCL11A (1:1000, Bethyl Laboratories, A300–380), HRP-conjugated goat anti-Rabbit secondary antibody (1:7500, Santa Cruz Biotechnology, sc-2004), rabbit anti-GFP (1:12,000, Life Technologies, A1112) and HRP-conjugated mouse anti-Actin (1:15,000, Abcam, ab49900). Protein abundance was visualized via the SuperSignal West Pico PLUS Chemiluminescent Substrate kit (Thermo Scientific).

SDS–PAGE was conducted using the standard protocol^{65 (link)}. The samples were denatured for 5 min at 95 °C in 1× Laemmli sample buffer (Bio-Rad) before loading on a 4–15% gradient precast protein gel (Bio-Rad). The electrophoresis was performed at a constant voltage of 200 V in SDS running buffer (Tris/Glycine/SDS, Bio-Rad) for about 30 min.
The gels used for western blotting were transferred onto nitrocellulose membrane. Membranes were blocked with 5% milk in TBS-T (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20) at 4 °C for 1 h or overnight. For His-tagged samples, membranes were incubated with 1:5,000 HRP-conjugated anti-6×His (no. MA1-21315-HRP, Invitrogen) in 1% milk in TBS-T for 1 h. Alternatively, membranes were incubated with 1:1,000 polyclonal rabbit anti-all3324 (anti-Cis1) or anti-all3325 (anti-Cis2) (GenScript) and 1:5,000 secondary HRP-conjugated goat anti-rabbit IgG (Abcam) in 1% milk in TBS-T for 1 h, respectively. Membranes were washed three times for 10 min in TBS-T between and after antibody incubations. Signals were detected using a chemiluminescent substrate (ECL, Amersham).