Western Blot Analysis of Protein Signaling

Whole cell extracts and nuclear cell extracts were prepared as described [25 (link)]. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham) by semi-dry blotting. For immuno-detection, the antibodies against Δ-Actin (C4, Santa Cruz), BIM (B7929, Sigma), FasL (G247-4, BD Pharmingen), ERK2 (sc-154, Santa Cruz), γH2AX (#05-164, Upstate), XPF (Ab-5, NeoMarkers), c-Fos (sc-52, Santa Cruz) were diluted 1:1000 in 5% non-fat dry milk/Tween-TBS. For western blot analysis with phospho-specific antibodies, cells were directly lyzed in 1 x SDS-PAGE sample buffer (Roti^®-Load 1, Carl Roth GmbH) and subsequently sonified. Rabbit phospho-specific antibodies (anti-p-cJun: #3270, anti-p-JNK: #4668P; anti-p-p38K: #4511P; anti-p-ERK1/2: #4370P) as well as non- phospho-specific antibodies (anti-cJun: #9165; anti-JNK: #9258P; anti-p38K: #9212P and anti-ERK1/2: #4695P) were purchased from Cell Signaling Technology (Boston, MA, USA) and diluted 1:1000 in 5% BSA/Tween-TBS. The secondary anti-rabbit or anti-mouse antibodies (Rockland) were diluted 1:2000 in 5% non-fat dry milk/Tween-TBS.