His tag

Proteins were extracted using RIPA lysis buffer (Millipore, Billerica, MA) containing protease inhibitor cocktail, 10 mM β-GP, and 5 mM Na₃VO₄. Protein from lysate was separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Millipore). The blots were probed overnight at 4 °C with primary antibodies against SEMA6A (custom manufactured antibody from GenScript), His-tag (Cat. #05–949; Millipore), β-actin (Cat. # ABT264; Millipore), and cleaved caspase-8 (Cat. #9496S; Cell Signaling). After incubation with the appropriate HRP-conjugated secondary antibodies, HRP activity was visualized by an enhanced chemiluminescence system (UVP, Upland, CA).

For western blotting, 10–30 μg of whole-cell extract, cytoplasmic protein, or mitochondrial extract were separated by 10% SDS-PAGE. Blots were probed with antibodies diluted 1:1000 in 5% skim milk against MATα1 (ab129176), PIN1 (07-091, Millipore), TOM20 (ab78547), DDK (TA50011-100, Origene), His-Tag (A00186-100, GenScript), OXPHOS (ab110413), CK2α (2656, Cell Signaling), CPT1α (12252, Cell Signaling), SDHα (5839, Cell Signaling), MCAD (ab110296), JNK (9252, Cell Signaling), pJNK (4668, Cell Signaling), p38 (9212, Cell Signaling), p-p38 (9211, Cell Signaling), GSK3β (9315, Cell Signaling), p-GSK3β (9336, Cell Signaling), methyl-lysine (ab23366), α-Tubulin (32-250022125, Invitrogen), β-actin (A3854 Sigma), and GAPDH (5174 Cell signaling Technology). HRP-linked secondary antibodies anti-rabbit (7074S Cell Signaling) and anti-mouse (7076S Cell Signaling) were diluted 1:5000 in 5% skim milk.