Irdye 680rd labeled anti mouse

Lysates were prepared from whole cells or purified mitochondria using RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton-X-100, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with 1× cOmplete protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Pierce). Total protein concentration was measured using the Bio-Rad Protein Assay kit. Proteins were resolved by SDS-PAGE and transferred onto a PVDF membrane. Protein-containing membranes were blocked with Odyssey Blocking Buffer (Licor) containing 0.1% Tween 20. Western blots were performed with either rabbit polyclonal anti-RUNX2 (Cell Signaling Technology #8486), monoclonal anti-COX1 (mitochondria-specific protein marker; Abcam, ab14705) or the monoclonal antibody KDEL (Lys-Asp-Glu-Leu: an ER-specific peptide marker; Abcam, ab12223). To normalize RUNX2 expression from whole cell lysates, mouse anti-β-actin antibodies were used (Abcam, ab6267). All primary antibodies were used at a 1:1000 dilution. Secondary antibodies (IRDye® 800CW-labeled anti-rabbit; IRDye® 680RD-labeled anti-mouse) (Licor) were used at a 1:10000 dilution following the manufacturer’s recommendation. Resulting protein bands were imaged and quantified using the Licor Odyssey software. To confirm successful purification of mitochondria, the ratio of COX1: KDEL was analyzed in lysates from whole cells and purified mitochondria samples.

Cell lysates were prepared with RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 % Triton-X-100, 0.1 % SDS, 0.5% sodium deoxycholate) supplemented with 1x cOmplete protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Pierce). Total protein concentration was measured using the Bio-Rad Protein Assay kit. Proteins were resolved by SDS-PAGE and transferred onto a PVDF membrane. Protein-containing membranes were blocked with Odyssey Blocking Buffer (Licor) containing 0.1 % Tween 20. Western blots were performed with the following rabbit polyclonal antibodies, all purchased from Cell Signaling Technology (anti-RUNX2 #8486; anti-PTEN #9552, anti-AKT #4691; anti-pAKT-T308 #2965; anti-pAKT-S473 #4060). For purposes of protein normalization, mouse anti-β-actin antibodies were used (Abcam ab6267). All primary antibodies were used at a 1:1000 dilution. Secondary antibodies (IRDye® 800CW-labeled anti-rabbit; IRDye® 680RD-labeled anti-mouse) (Licor) were used at a 1:10000 dilution following the manufacturer’s recommendation. Resulting band intensity was calculated and quantified using the Licor Odyssey software.

Cell lysates containing protein (10 μg) were resolved by SDS-PAGE and transferred onto PVDF membranes. Immunoblotting was carried out using a rabbit polyclonal DNM3 antibody (LSBio LS-C409118) or mouse β-actin antibody (Abcam ab6267). Primary antibodies were used at 1:1000 dilution. Secondary antibodies (IRDye 800CW-labeled anti-rabbit; IRdye 680RD-labeled anti-mouse; LI-COR Biosciences) were used at 1:10,000 dilution following the manufacturer’s recommendation. Immunoblot images were generated using the LI-COR Odyssey near-infrared imager. Protein band density was quantified using LI-COR software.