Ez run pre stained protein ladder

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotics. iSLK cells were cultured with the same medium plus 450 µg/mL G418 and 1 µg/mL puromycin [34 (link)]. The generations of iSLK.BAC16, iSLK.BAC16-ORF45 ΔC19, and iSLK.BAC16-ORF45 ΔC19R cells were described previously [20 (link),35 (link),36 (link)]. These cells were cultured similarly to iSLK with the addition of 400 µg/mL hygromycin B. Anti-HA, anti-FLAG M2, anti-GST antibodies, and EZview Red anti-FLAG M2 resin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-USP7 antibody was purchased from Bethyl Laboratories (Montgomery, TX, USA). Antibodies that detect ORF45 were generated as previously described [11 (link),37 (link)]. Monoclonal antibodies against ORF26, ORF33, and ORF38 were generated as described [21 (link)]. Protein-G beads were purchased from Thermo Fisher Scientific (Waltham, MA, USA). EZ-Run Pre-Stained Protein Ladder was purchased from Fisher Scientific (Pittsburgh, PA, USA). TAT-C19 and TAT were synthesized by Biomatik (Wilmington, DE, USA).

CD4⁺ T cells were grown in Treg-inducing conditions with either B7x-Ig or Control-Ig stimulation as described above in 6-well cell culture dishes. After 24 h, the medium was aspirated and the cells were lysed with RIPA buffer (1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris, 150 mM NaCl) supplemented with Pierce protease and phosphatase inhibitor cocktail (Thermofisher). Total protein in the lysates were measured by BCA protein assay (Thermofisher). Equal total protein volumes were run on ExpressPlus 4–12% gels (Genscript) and wet-transferred to nitrocellulose membranes (Thermofisher). EZ-Run Prestained protein ladder (Fisher Scientific) was used for monitoring transfer efficiency and approximating molecular weights. The membranes were blocked with 5% milk in TBST for 1 h at room temperature, then probed with primary antibodies anti-Akt, anti-p-Akt, anti-Foxo1, anti-p-Foxo1 (CST) overnight at 4 °C, and then stained with secondary anti-rabbit HRP (CST) for 1 h at room temperature. Blots were detected with Pierce ECL substrate kit (Thermofisher) and visualized with the ChemiDoc imaging system (Bio-Rad).