Anti sae2

Cells were lysed in radioimmune precipitation assay buffer (150 mM NaCl, 0.5% sodium deoxycholate, 50 mM Tris-HCl pH 8.0, 0.1% SDS, 0.1% Nonidet P-40) supplemented with protease inhibitors (Roche, Boulogne-Billancourt, France) and 5 mM N-ethylmaleimide (Sigma). Proteins were separated on SDS/PAGE gels, transferred to nitrocellulose membranes (Amersham Biosciences, Velizy-Villacoublay, France), blocked with 5% non-fat milk in PBS containing 0.1% Tween-20. Membranes were then probed overnight at 4°C with the relevant primary antibodies: anti-SUMO1 (Sigma), anti-SUMO2/3 (Sigma), anti-LC3 (Sigma), anti-SAE1 (Abcam, Paris, France), anti-SAE2 (Abcam), anti-UBC9 (Abcam), anti-PIAS3 (Cell Signaling Technology, Saint Quentin Yvelines, France), anti-p62 (Cell Signaling Technology), anti-β-actin (Cell Signaling Technology), and anti-GAPDH (Cell Signaling Technology). After washes, membranes were incubated with the appropriate HRP-conjugated secondary antibodies (Cell Signaling Technology), and blots were detected using the Enhanced Chemiluminescence Detection kit (Amersham Biosciences, Charfent, UK).

Cells were lysed in RIPA buffer plus protease inhibitors (leupeptin 1 μg/mL, aprotinin 1 μg/mL, PMSF 100 μg/mL and EDTA 1 mM) and 0.5 mM N-ethylmaleimide (NEM). Equal amount of total proteins were resolved by SDS-PAGE under reducing conditions, and immunoblotted with the indicated antibodies: anti-UBC9 (Abcam), anti-SUMO1 (Sigma-Aldrich), anti-SUMO2/3 (Abcam), anti-RanGAP1 (Santa Cruz Biotechnology), anti-p53 (DO-1; Santa Cruz Biotechnology), anti-pRb (Santa Cruz Biotechology), anti-SAE1 (Abcam), anti-SAE2 (Abcam), anti-Ubiquitylated proteins (FK1, Biomol), anti-LC3 (Novus Biologicals), anti-p62 (2C11, Abnova), anti-EGFR (kindly provided by S. Sigismund, Fondazione Istituto FIRC di Oncologia Molecolare, IFOM, Milan [86 (link)]), anti-ATG5 (Cell Signaling Technology), anti-pRb (BD Pharmingen). Anti-GAPDH (Abcam), anti-tubulin (Sigma-Aldrich), or anti-vinculin (Santa Cruz Biotechnology) antibodies were used as loading control. Membranes were then incubated with the appropriate IR Dye-conjugated or horseradish peroxidase (HRP) secondary antibodies (Licor), and scanned with a LI-COR Odyssey Imager or acquired with Chemidoc (Bio-rad), respectively. The intensities of the protein bands were quantified using ImageJ software (Rasband W.S., ImageJ, U. S. National Institutes of Health), and standardized to the housekeeper levels.