Figure 4.

MKRN1 induces the degradation of p14ARF. A) MKRN1 induces the degradation of p14ARF in a dose-dependent manner. H1299 cells were transfected with mixtures of plasmids expressing p14ARF/FLAG and HA/MKRN1 with increasing concentrations, or with GFP. The cell lysates were detected with anti-FLAG, HA, and GFP antibodies. B) MKRN1 decreases the half-life of p14ARF. H1299 cells were transfected with mixtures of plasmids expressing p14ARF/FLAG, HA/MKRN1, or GFP followed by cycloheximide (CHX) treatment before harvest. The lysates were immunoblotted using antibodies against FLAG, HA, and GFP. C) Exogenous MKRN1 induces degradation of endogenous p14ARF. HeLa cells were transfected with increasing amounts of HA/MKRN1 plasmids. The cell lysates were immunoblotted with anti-p14ARF, HA, and actin antibodies. D) Exogenous MKRN1 reduces the stability of endogenous p14ARF. HeLa cells were transfected with mock vector or plasmid expressing HA/MKRN1 followed by CHX treatment before harvest. The cell lysates were immunoblotted with anti-p14ARF, HA, and actin antibodies. E) MKRN1 depletion stabilizes endogenous p14ARF. HeLa cells were transfected with control, MKRN1 #5, or MKRN1 #6 siRNAs and lysed after 48h. WCL were immunoblotted with anti-p14ARF, MKRN1, and actin antibodies. F) MKRN1 ablation does not affect p14ARF mRNA levels. HeLa cells were treated as above and analyzed by RT-PCR using primers specific for p14ARF, MKRN1, and GAPDH. G) MKRN1 depletion increases stability of endogenous p14ARF. HeLa cells were transfected with control and MKRN1 siRNAs followed by treatment with CHX and were then lysed and immunoblotted with anti-p14ARF, MKRN1, and actin antibodies. The graphs indicate the relative amounts of p14ARF protein compared with the levels of actin or GFP in the western blot. The expression of each protein was analyzed using the Image J program. MKRN1 = Makorin ring finger protein 1; p14ARF = p14 alternative reading frame; CHX = cycloheximide; WCL = whole cell lysates.