Anti ulk

Cells were harvested and lysed with 20 mM Tris, 135 mM NaCl, 10% glycerol, 1% NP40, pH 6.8. Protein content of cell lysates was measured using Pierce BCA Protein Assay (Thermo Scientific, 23225). During each procedure equal amounts of protein were used. SDS-PAGE was done by using Hoefer miniVE (Amersham). Proteins were transferred onto Millipore 0.45 μM PVDF membrane. Immunoblotting was performed using TBS Tween (0.1%), containing 5% non-fat dry milk for blocking membrane and for antibody solutions. Loading was controlled by developing membranes for GAPDH or dyed with Ponceau S in each experiment. For each experiment at least three independent measurements were carried out. The following antibodies were applied: antiLC3B (SantaCruz, sc-16755), anticaspase-3 (SantaCruz, sc-7272), antiPARP (Cell Signaling, 9542S), antiULK555P (Cell Signaling, 5869S), antiULK (Cell Signaling, 8054S), antip70S6P (Cell Signaling, 9234S), antip70S6 (SantaCruz, sc-9202), anti4EBP1P (Cell Signaling, 9459S), anti4EBP1 (Cell Signaling, 9644S), antiGADD34 (SantaCruz, sc-8327), antieiF2αP (SantaCruz, sc-9721L), antieiF2α (SantaCruz, sc-9722S), antip62 (Cell Signaling, 5114S) and antiGAPDH (Santa Cruz, 6C5), HRP conjugated secondary antibodies (SantaCruz, sc-2020 and Cell Signaling, 7074S, 7076S).

Antibodies against microtubule-associated protein light chain 3 (LC3) and α-tubulin were obtained from Sigma Chemical Co. (St Louis, MO, USA) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), respectively. Anti-p21^Waf1/Cip1, anti-p27^Kip1, anti-cyclin D1, anti-caspase-3, poly (ADP-ribose) polymerase (PARP), anti-c-jun-N-terminal kinase (JNK), anti-phospho-JNK (Thr183/Tyr185), anti-adenosine monophosphate-activated protein kinase (AMPK), anti-phospho-AMPK (Thr172), anti-ULK, anti-phospho-ULK (Ser555), anti-c-jun, anti-phospho-c-jun (Ser73), anti-ERK1/2, anti-phospho-ERK1/2 (Thr202)/Tyr204), anti-p38, anti-phospho-p38 (Thr180/Tyr182), anti-p53, anti-phospho-p53 (Ser15), anti-Bcl-2, anti-p62/SQSTM1, and anti-Beclin-1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Propidium iodide (PI), Ribonuclease A (RNase A) from bovine pancreas, monodansyl cadaverine (MDC), 2′7′-dichlorofulorescein diacetate (DCF-DA), Bafilomycin A1 from Streptomyces griseus, and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical. SP600125, SB203580, PD98059, and N-acetyl-L-cysteine (NAC) were obtained from Calbiochem (San Diego, CA).