Anti strep tag 2

Cells were lysed in CETi lysis buffer (TransLab, Daejeon, Korea) and the lysate was separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After transferring the proteins onto polyvinylidene fluoride membranes (Merck), the membranes were blocked and incubated with primary antibodies overnight at 4 °C. After washing, horseradish peroxidase (HRP)-conjugated secondary antibodies were added. Bound antibodies were visualized using Immobilon Forte Western HRP substrate (Merck) and imaged using EZ-Capture II (ATTO Corporation, Tokyo, Japan). The following antibodies were used: anti-phospho-SAPK/JNK (Cat# 9255), anti-SAPK/JNK (Cat# 9252), anti-phospho-p44/42 MAPK (Cat# 4370), anti-p44/42 (Cat# 4695), anti-phospho-p38 MAPK (Cat# 4511), anti-p38 MAPK (Cat# 54470), anti-phospho-IκB (Cat# 2859), anti-IκB-alpha (Cat# 4812), anti-phospho-NF-κB p65 (Cat# 3033), anti-COX1 (Cat# 9896), anti-COX2 (Cat# 12282), anti-iNOS (Cat# 13120), anti-Bim (Cat# 2933), anti-phospho-Bad (Cat# 5284), anti-Bak (Cat# 12105), anti-caspase-3 (Cat# 9665), anti-caspase-6 (Cat# 9762), anti-caspase-7 (Cat# 8438), anti-caspase-9 (Cat# 9508), HRP-linked anti-mouse IgG (Cat# 7076), HRP-linked anti-rabbit IgG (Cat# 7074),(all from CST, Danvers, Massachusetts, USA), and anti-Strep-tag II (Abcam, Cat# ab76949).

Proteins were extracted from day 2 cultures; cells were harvested by centrifugation at 5000 rpm, 4 °C, for 10 min. The pellet was washed in 2 ml PBS and recollected by centrifugation, 5000 rpm, 4 °C, for 10 min, and resuspended in 200 µl PBS with Protease Arrest (GBioscience). This mixture went through freeze–thaw process and was disrupted by acid-washed 425–600 μm-diameter glass beads (Sigma-Aldrich) using a Precellys-24 Beadbeater (Bertin Instruments), program 3 × 30 s. Centrifugation was performed twice at 12,000 rpm, 4 °C, 1 min each, to get a clean supernatant-containing soluble proteins. Protein concentration was determined by the DC protein assay (Bio-Rad). 5 μg soluble proteins from each strain were separated by SDS-PAGE, using Mini-PROTEAN TGX TM gels (Bio-Rad), and transferred to PVDF membrane (Bio-Rad). Anti-Strep-tag II (abcam) antibody was used to detect Strep-tagged Kivd through the standard techniques, while anti-ATPase western immunoblot was done on the same samples as loading control. This western immunoblot was repeated three times for determining the relative Kivd expression level in all strains.

Human 293 Flp-In™ T-REx™ (293T) (Invitrogen) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in the presence of 5% CO₂. To prepare stable cell lines with inducible expression of the fusion proteins, 293T cells were grown to 70% confluency in 6-well plates format and co-transfected with 300 ng of the corresponding expression vector (pcDNA5/FRT/TO™, Invitrogen, MAC-tag-N or HA-Strep II tag) and 2.7 μg of pOG44 vector (Invitrogen) (ratio 1:9) using 5 μl of TurboFect reagent (Invitrogen) following manufacture's protocol. One day after transfection, cells were transferred to a 150 mm dish and selected in 60 μg/ml of hygromycin B until individual clones were formed. Doxycycline (dox)-inducible expression of the tagged proteins were confirmed by western blot with anti-Flag antibodies (Sigma, 1:5000) or anti-Strep tag II (Abcam, 1:2000) and individual clones were selected.