DZIP1 Protein Stability Assay

HEK293T cells were seeded at 5 × 10⁵/well on six-well plate. On the next day, cells were co-transfected with wild-type DZIP1-Flag or DZIP1^C585W-Flag and CBY1-HA constructs using FuGENE HD transfection Reagent (Promega, Cat No: E2311). Cycloheximide was added 48 hours post transfection at concentration of 100 ng/mL. Cells were lysed with RIPA buffer at 0, 4, 8, 16, 32 hours after cycloheximide treatment. Dzip1^S14R/+ and wild-type MEFs were seeded at 2 × 10⁵/well on six-well plate and treated with cycloheximide at concentration of 100 ng/mL the next day. Cell lysates were harvested using RIPA buffer at 0 and 48 hours post cycloheximide treatment. Immunoblotting was performed as described previously.^{11 (link)} Transfections were performed in triplicate and repeated a minimum of three times. Primary antibodies used for western blot were: α-mouse Flag M2 (Sigma), α-rabbit HA (Sigma), α-rabbit CBY1(Protein tech), α-rabbit DZIP1 (Protein tech), α-mouse actin (Millipore). HRP-conjugated secondary antibodies were purchased from Sigma.