Western blot analysis of Ubiquitin was performed on HeLa cells either left untreated (right panel) or treated with 10uM of MG132 proteasome inhibitor (left panel) for 2 hours at 37C. Whole cell lysates were prepared using IP Lysis buffer (Product # 87787), and 10 µg of the lysates along with 5 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) were loaded onto a Novex® 10-20% Tricine Gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288) and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for 1 hour at room temperature. Ubiquitin was detected at ~8.5 kDa after probing with a Ubiquitin polyclonal antibody (Product # PA1-187) at a dilution of 1:1000 in StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) overnight at 4C on a rocking platform, washing in TBST, and probing with an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:40,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). NOTE: In addition to detecting ubiquitin monomer, this antibody also detects poly-ubiquitinated proteins at various molecular weights.