Anti uqcrb

Soluble proteins were harvested from cells using a SDS lysis buffer comprising 50 mM Tris HCl with a pH of 6.8, containing 10% glycerol, 2% SDS, 10 mM dithiothreitol, and 0.005% bromophenol blue. Equal concentrations of proteins were separated by 11% or 12.5% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Blots were then blocked and immunolabeled overnight at 4 °C with the following primary antibodies: anti-actin (Abcam, ab6276), LAMP1 (Abcam, ab24170), anti-LC3B (Cell Signaling Technology, 2775), anti-Myc (Abcam, ab18185), anti-p62 (BD Biosciences), anti-UQCRB (Abcam, ab190360). Immunolabeling was visualized using an enhanced chemiluminescence kit (Amersham Life Science, Chalfont, UK), according to the manufacturer’s instructions. Images were quantified using Image Lab software (Bio-Rad, Hercules, CA, USA). Actin and GAPDH were used as internal controls. All band intensities were proportional to the amount of target protein on the membrane as determined by the linear range of detection. Images were quantified using Image LabTM software (Bio-Rad).

Tissues and cells were lysed with RIPA buffer containing Halt Protease/Phosphatase inhibitors (Thermo Fisher Scientifics, Pittsburgh, PA). The primary antibodies used in this study were as follows: anti-phospho (Ser563)-HSL (#4139), anti-phospho (Ser660)-HSL (#4126), anti-HSL (#4107), anti-pPKA Substrate (#9621), and anti-phospho (Ser133)-CREB (#9198) (all from Cell Signaling Technology); anti-SLC22A3 antibody (#ab191446), anti-ATP5A (#ab176569), anti-COX7B (#ab140629), anti-UQCRH (#ab134949), anti-UQCRB (#ab190360), and anti-beta Actin (ab8226) (all from Abcam); anti-UCP1 (#GTX112784) (Genetex); and anti-PGC1α (#20658) (Proteintech Group Inc).