Fig. 5.

p16^INK4Aaggregates in live cells in response to oxidation.

(A) Set up for the Filter Trap assay for the detection of aggregates in cell lysates.

(B) Results for the Filter Trap assay. Note that the majority of p16^INK4A in the pellet is in the form of S-S-linked homodimers after lysis. p16^INK4A, but not p16^INK4AC72A, was trapped on the filter membrane upon treatment with 200 μM diamide and trapping was prevented by pretreatment with DTT. Equal amounts of p16^INK4A and p16^INK4AC72A were used as input for the filter trap assay. Note that the input for the Filter Trap assay runs mainly as a S–S-dependent dimer after boiling in non-reducing SDS-PAGE sample buffer. R: reducing, NR: non-reducing.

(C) Aggregates of p16^INK4A peak around 30  min and are then cleared over time.

(D) Endogenous p16^INK4A also forms S–S-linked dimers that (partially) form aggregates in the insoluble pellet fraction. Note that also mixed S–S-linked dimers of endogenous and overexpressed p16INK4A can be observed in lanes where FLAG-p16INK4A was transfected. n.i.: IP with non-immune serum as a control. p16(r): rabbit anti-p16INK4A, p16(m): mouse anti-p16INK4A, e.v.: empty vector, Red: reducing, Non-Red: non-reducing. All experiments were performed at least 3 times. Representative experiments are shown.