Mini protean 4 20 gradient gels

Indicated samples were fractionated by SDS-PAGE gel electrophoresis in mini-PROTEAN 4–20% gradient gels (Bio-Rad). Protein was transferred to PVDF according to the manufacturer’s protocols (Bio-Rad). After blocking with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.5% Tween 20) for 1 h, the membrane was probed with primary antibodies, rabbit anti–14-3-3 pan (1:200; 51-0700; Invitrogen) and mouse anti-Dlg (1:1,000; 4F3; Developmental Studies Hybridoma Bank). Secondary HRP-conjugated antibodies, goat anti-rabbit (1:10,000; 111-035-003; Jackson ImmunoResearch) and goat anti-mouse (1:10,000; A10668; Invitrogen), were used for detection using SuperSignal West Dura (Thermo Fisher Scientific). Blots were exposed to film (Hyperfilm MP; GE Healthcare) to generate images using a film processor (X-OMAT 200A; Kodak).

Samples for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were prepared from ca. 5 μg protein mixed with 2× Laemmli buffer from Bio-Rad (product number 1610737) supplemented with 25 mM DTT. They were incubated at 90 °C for 10 min before loading them on precast Mini-Protean 4–20% gradient gels from Bio-Rad (product number 4568093). The molecular size marker was from Bio-Rad (product number 64060948). SDS-PAGE was carried out using a voltage of 180 V. The gels were stained by AcquaStain from Lubio science (product number LU0010000).