Mabe343

Day 1 adult N2 worms were bleached, and eggs were allowed to hatch in S-complete by shaking overnight. On the next day, 12,000 L1 worms were seeded in a 15 cm plate containing a total volume of 30 mL S-complete with 6 mg/mL OP50 bacteria, 50 μg/mL carbenicillin, and 0.1 μg/mL amphotericin B. Six mL of 0.6 mM FUDR were added to worms at L4 stage in each plate. 100 μΜ translation inhibitor was added to worms 2 hr after adding FUDR. After 12 hr, worms were transferred into a 15 mL corning tube containing a total volume of 5 mL S-complete with 750 µL 6 mg/mL OP50 bacteria, 0.5 mg/mL puromycin, and 100 μΜ translation inhibitors. After rotating the corning tubes for 4 hr, worms were collected into 2 mL cryotubes by washing them with M9 once and with cold PBS three times. Worms were flash-frozen in liquid nitrogen and subsequently broken with a beak mill homogenizer (Fisherbrand). Protein concentrations were determined by the Bradford protein assay. 50 mg protein from each sample was loaded for western blot analysis using antibodies against puromycin (Millipore, MABE343) and GAPDH (Proteintech, 10494-1-AP). Antibodies were diluted 1:5000 in 5% non-fat milk in TBST.

ABT-737 was prepared using the same method as described in the WST-1 assay. Primary antibodies against the following targets were purchased from Cell Signaling Technology: p-FAK(#8556), FAK (#7143), p-AKT (#2965), AKT (#9272), p-PKCα (#9375), PKCα (#2056), GAPDH (#2118), Bcl-2 (#4223), Bcl-xL (#2764), Mcl-1 (#5453), Bim (#2933), Bax (#5023), and PARP (#9542). Antibodies against puromycin (Millipore #MABE343) and furin (ProteinTech #18413-1-AP) were also obtained. An anti-PE38 antibody capable of binding to both intact and cleaved PE38 toxins was kindly provided by Dr. Ira Pastan’s laboratory at National Institutes of Health (NIH). All experiments were repeated at least 2 times.

The following antibodies were used in this study:
Primary antibodies:

PSD-95, Abcam ab19257 (WB 1:2000, IF 1:200)

MAP2, Synaptic System 188004 (IF 1:300)

β-Actin, Sigma A5441 (WB 1:15,000)

Puromycin, Millipore MABE343 (WB 1:5000, IF 1:300)

Histone H3, Proteintech 17168-1AP (WB 1:2000)

Synaptophysin, Abcam ab32127 (WB 1:50,000)

Mpc1, Sigma-Aldrich HPA045119 (WB 1:1000)

Glutamate dehydrogenase Cell Signaling 12793 (WB 1:1000)

GFAP Cell Signaling 367 (WB 1:3000).

Secondary antibodies:

Goat anti-rabbit (HRP-conjugated), Bio-Rad 170-6515 (1:10,000)

Goat anti-mouse (HRP conjugated), Bio-Rad 172-1011 (1:10,000)

Alexa Fluor 488 goat anti-rabbit, Invitrogen A11034">A11034 (1:1000)

Alexa Fluor 555 goat anti-mouse, Invitrogen A21424">A21424 (1:1000)

Alexa Fluor 647 goat anti-guinea pig, Invitrogen A21450">A21450 (1:300).