Anti mouse or anti rabbit igg secondary antibodies

SDS-PAGE or BN-PAGE gels were transferred to polyvinylidene difluoride (PVDF) membrane and probed with primary antibodies against ATAD3^{28 (link)}, ANT3 (Abcam anti-SLC25A6, ab154007), SDHA/complex II 70 kDa subunit (for SDS-PAGE: Molecular Probes, A-11142; for BN-PAGE: Abcam, ab14715), Porin/VDAC1 (Abcam ab14734), Total OXPHOS Human WB Antibody Cocktail (Abcam, ab110411), complex I subunit NDUFA9 raised in-house^{93 (link)}, ATAD3A (Abnova, H00055210-D01), ATAD3B (Abnova, H00083858-B01P), Tim23 (BD Bioscience, 611223), V5 (Invitrogen, R960–25), Core1 (ThermoFisher, 459140) and Cox4 (Abcam, ab110261). Blots were incubated with anti-mouse or anti-rabbit IgG secondary antibodies (GE Healthcare) and developed with Clarity Western ECL Substrate (Bio-Rad Laboratories).

Cells were trypsinized, centrifuged and washed with PBS before lysing with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, Boston BioProducts) containing protease and phosphatase inhibitor cocktails (Calbiochem). Lysates were sonicated twice and incubated on ice for 30 mins, then centrifuged at 16,000 g for 10 min. Protein concentration was quantified using a Pierce-BCA assay with a standard curve. Protein separation by gel electrophoresis using 4%–20% agarose gels and transferred to PVDF. Membranes were immunoblotted for 1 hour at room temp or overnight at 4°C with primary antibodies, then washed and probed with anti-mouse or anti-rabbit IgG secondary antibodies (GE Healthcare) for 1 hour at room temperature. Chemiluminescence was detected using X-ray films. Extracellular Cytokines were analyzed using the Human Cytokine Array Kit performed per manufactures instructions (R&D Systems). Parental and palbociclib-resistant cells were seeded in parallel and medium harvested after 24 hours for processing.