Western Blot Analysis of STAC3 in HEK Cells

Proteins isolated from the three HEK cell lines were prepared as previously described (Campiglio and Flucher, 2017 (link)). Briefly, cells plated in 100-mm dishes were trypsinized after 48 h in culture. Cells were lysed in radioimmunoprecipitation assay buffer with a pestle and left on ice for 30 min. The lysates were then centrifuged for 10 min. The protein concentration was determined using a BCA assay (cat. #23250; Pierce). 20 µg of protein samples were loaded on a NuPage gel (4–12% polyacrylamide, cat. #NP0321; Invitrogen) and separated by SDS-PAGE at 160 V. The protein samples were then transferred to a PVDF membrane at 25 V and 100 mA for 3 h at 4°C with a semidry blotting system (Roth). The membrane was then cut and incubated with rabbit anti-STAC3 (1:2,000; cat. #20392-1; Proteintech; RRID:AB_10693618) or mouse anti-GAPDH (1:100,000; cat. #sc-32233, Santa Cruz Biotechnology; RRID:AB_627679) antibodies overnight at 4°C and then with HRP-conjugated secondary antibody (1:5,000; Pierce) for 1 h at room temperature. The chemiluminescent signal was developed with ECL Supersignal WestPico kit (cat. #34579; Thermo Fisher Scientific) and detected with ImageQuant LAS 4000.