Total plin5

Cell lysates (30–50 μg protein) were separated on 10–12% tricine gels using a Mini-Protean II cell (Bio-Rad lab, Hercules, CA) system at constant amperage (30 mA per gel) for about 3 hrs. Proteins were then transferred onto PVDF membranes at constant voltage (90 V) for 1.5 hrs. Blots were stained with Ponceau S to confirm uniform protein loading (Aldridge et al., 2008 ; Willenborg et al., 2005 (link)) before blocking in 5% BSA in TBST (10 mM Tris-HCl, pH 8, 100 mM NaCl, 0.05% Tween-20) for 1 hr. Blots were incubated with specific poly- or monoclonal antibodies overnight and were developed with IRDye 800CW (LI-COR) or IRDye 680RD (LI-COR) secondary antibodies. To visualize the bands of interest, blots were scanned using the LI-COR Odyssey imaging system (Lincoln, NE). Protein bands were quantitated by densitometric analysis after image acquisition using NIH Scion Image to obtain relative protein levels expressed as integrated density. All values were normalized to β-actin expression or PonceauS staining. Antibodies were purchased or obtained from the following sources; Total-Plin5 (Progen; Heudelberg, Germany), Histone H3, SIRT1, Acetylated Lysine (Cell Signaling Technologies; Danvers, MA), PGC-1α (MilliporeSigma; Burlington, MA), phospho-PLIN5 (NeoBioLab targeting; Cys-LARRGRRW(pS)VELK), PLIN2 [Barbara Atshaves developed in (Atshaves et al., 1999 (link))].

Cell lysates (30–50 μg protein) were separated on 10–12% tricine gels using a Mini-Protean II cell (Bio-Rad lab, Hercules, CA) system at constant amperage (30 mA per gel) for about 3 h. Proteins were then transferred onto PVDF membranes at constant voltage (90 V) for 2 h. Blots were stained with Ponceau S to confirm uniform protein loading^{91 (link),92 (link)} before blocking in 5% BSA or 7% non-fat milk in TBST (10 mM Tris-HCl, pH 8, 100 mM NaCl, 0.05% Tween 20) for 1 h. Blots were incubated with specific poly- or monoclonal antibodies overnight and were developed with IRDye 800CW (LI-COR) or IRDye 680RD (LI-COR) secondary antibodies. To visualize the bands of interest, blots were scanned using the LI-COR Odyssey imaging system (Lincoln, NE). Protein bands were quantitated by densitometric analysis after image acquisition using NIH Scion Image to obtain relative protein levels expressed as integrated density. All values were normalized to Ponceau S staining.^{93 (link)–96 (link)} Antibodies were purchased or obtained from the following sources; Total-Plin5 (Progen; Heudelberg, Germany), OXPHOS ACADVL, ACADM, ACAA2, CS, ACO1, IDH3, SUCLG1, FH, MDH2, Catalase, COXIV, Calreticulin, VAP-B, MIGA2, VAPB, SLC25a1, FAS, ACSL1, ACSL3, ACSL 5, SCD1, FADS6, AGPAT2, CPT1α, BIP, Calreticulin, and PLIN2 [Barbara Atshaves developed in^{72 (link)}].