Ubc9 h 81

Protein fractions were separated by SDS-PAGE (8% to 15% depending on the experiment) and transferred onto nitrocellulose. Membranes were blocked with 1% BSA in PBS-Tween 0.1% (PBS-T) for 1 h, followed by incubation with primary antibodies in blocking buffer overnight at 4°C. Membranes were washed three times with PBS-T for 10 min and then incubated with secondary antibodies conjugated to horseradish peroxidase in blocking buffer for 1 h. Membranes were washed four times with PBS-T for 15 min, and signals were detected by enhanced chemiluminescence (Santa Cruz sc-2048 or Biorad Clarity 1705061). Antibodies to SUMO1 (FL-101, 1:1000 dilution), SUMO2 (FL-103, 1:2000 dilution), Ubc9 (H-81, 1:500 dilution), His (H-15, 1:500 dilution), p53 (DO-1, 1:5000 dilution), myc (sc-40; 1:2000 dilution) and actin (C-11, 1:2000 dilution) were from Santa Cruz. Antibodies to PML (A301-167A, 1:2000 dilution), GST (A190-122A; 1:10,000) and FLAG (S190-102, 1:10000 dilution) were from Bethyl. Anti-FLAG (F1804, 1:10000 dilution) was from SIGMA and anti-ICP0 (H1A027, 1:5000 dilution) was from Virusys. RNF4 antibody (a kind gift from Ronald T. Hay; [109 (link)]) and was used at 1:5000 dilution.

Antibody against KHSRP (#A302–021) was from Bethyl Laboratories, Inc. (Montgomery, UK). Tubulin-Alpha mouse Mcab (#66031–1-Ig), His-Tag mouse antibody (#66005–1-Ig), GST mouse antibody (#66001–1-Ig) were from Proteintech™. GAPDH (#ab37168), SUMO1 (Y299, #ab32058) antibodies were from Abcam. Mouse antibodies against Flag (M2, #F1804), HA (16B12, #MMS-101P) were from Sigma. Rabbit antibodies against HA (Y-11, #sc805), Ubc9 (H-81, #sc-10,759) and mouse antibody against Lamin B1 (8D1, #sc-56,144) were from Santa Cruz Biotech. Rabbit antibody against SUMO1 (C9H1, #4940S) was from Cell Signal Technology. Alexa Fluor® 568 (Rabbit, #A11011), 488 (Rabbit, #A11008), 488 (Mouse, #A11001) were from Invitrogen.
Protein G Plus/Protein A agarose suspension (#IP05) were purchased from Calbiochem. Puromycin (#P8833), hydrogen peroxide (H₂O₂) solution (#H1009), insulin (#I-5500), LY294002 (#L9908) and EGF (#E9644) were from Sigma.