Sumo protein assay

Immunoblots were performed as previously described [93 (link)]. Briefly, cell lysates were harvested with sumo lysis buffer with protease inhibitors (cOmplete, Roche). Quantitation of protein concentration was conducted with a sumo protein assay (Biorad). The lysates were separated using a 10% polyacrylamide gel and then transferred onto a nitrocellulose membrane. The membranes were subsequently blocked with 5% milk consisting of .1% Tween 20 and 1X PBS for one hour. Membranes were then incubated with primary antibody overnight. The following day the antibodies were removed and the membrane was washed with wash buffer (1X PBS, 0.1% Tween 20) three times for 5 minutes. The membrane was then incubated with secondary antibody suspended in 5% milk for one hour, before washing with wash buffer three times for 10 minutes before treatment with ECL (Thermofisher) and imaging. Image Studio Lite software was used to quantify levels of proteins of interest relative to loading controls tubulin or actin in certain figures.

Protein samples were harvested from cells in culture as previously described [86 (link)], and from frozen primary tumor tissue by homogenizing tissue with the Covaris Cryoprep Pulverizer as previously described [6 (link)]. Cell and tissue lysates were harvested in Sumo lysis buffer including protease inhibitors (Roche) and phosphatase inhibitors (Sigma) as described previously [87 (link)]. Protein concentration was determined using the Sumo protein assay (Biorad), and proteins were separated in SDS-10% and -15% polyacrylamide gels and transferred onto nitro-cellulose membranes. Membranes were blocked in PBS containing 5% milk, and 0.1% Tween 20 solution. Membranes were then incubated with primary and secondary antibodies listed in Table 3. Image Studio Lite software was used to quantify levels of NFATc2, Z, R, and BMRF1 relative to loading control actin in Fig 11 [20 (link)].

Cell lysates were harvested in Sumo lysis buffer including protease inhibitors (Roche) as described previously [96 (link)]. Protein concentration was determined using the Sumo protein assay (Biorad), and proteins were separated in SDS-10% polyacrylamide gels and then transferred onto a nitro-cellulose membrane. Membranes were blocked in PBS containing 5% milk, and 0.1% Tween 20 solution. Membranes were then incubated in the following primary antibodies: anti-Z (Santa Cruz, product # sc-53904, 1:250), anti-BMRF1 (Millipore, product # MAB8186, 1:3,000), anti-R rabbit polyclonal antibody directed against the R peptide (peptide sequence EDPDEETSQAVKALREMAD, 1:2,500), anti-KLF4 (Cell Signaling, product # 4038, 1:1,000), anti-BLIMP1 (Cell Signaling, product # 9115, 1:1,000), anti-β-actin (Sigma, product # A5441,1:5,000), anti-tubulin (Sigma, product # T5168, 1:2,000), and anti-involucrin (Sigma, product # I9018, 1:3000). The secondary antibodies used were horseradish peroxidase (HRP)- labelled goat anti-mouse antibody (Fisher Scientific, 1:5,000) and HRP- labeled anti-rabbit antibody (Fisher scientific, 1:5,000).