Quantitative Immunoblotting Analysis of SUMOylation

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.

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Mattoscio D., Casadio C., Miccolo C., Maffini F., Raimondi A., Tacchetti C., Gheit T., Tagliabue M., Galimberti V.E., De Lorenzi F., Pawlita M., Chiesa F., Ansarin M., Tommasino M, & Chiocca S. (2017). Autophagy regulates UBC9 levels during viral-mediated tumorigenesis. PLoS Pathogens, 13(3), e1006262.

Publication 2017

anti-UBC9 anti-SUMO1 anti-SUMO2/3 anti-p53 (DO-1; anti-pRb anti-SAE1 anti-SAE2 anti-LC3 anti-p62 (2C11, anti-ATG5 Anti-GAPDH anti-tubulin anti-vinculin Chemidoc

Antibodies Aprotinin Biologicals Buffer Cells Edta Egfr Ethylmaleimide Gapdh Horseradish peroxidase Leupeptin Membranes Novus Protease inhibitors Protein Rangap1 Ripa Sds page Sumo1 Tubulin Vinculin

Corresponding Organization : Heidelberg University

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Variable analysis

independent variables

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)

dependent variables

Expression levels of proteins: UBC9, SUMO1, SUMO2/3, RanGAP1, p53, pRb, SAE1, SAE2, Ubiquitylated proteins, LC3, p62, EGFR, ATG5, pRB

control variables

Equal amount of total proteins were resolved by SDS-PAGE under reducing conditions
GAPDH, tubulin, or vinculin antibodies were used as loading control

positive controls

None specified

negative controls

None specified

Annotations

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