Sc 271028

EL4 cells transfected overnight with pEGFP-C3-siOCILRP2 were stimulated with anti-CD3/CD28 antibodies. For the other two groups, EL4 cells were stimulated with or without anti-CD3/CD28 antibodies. Cell lysates were prepared in SDS sample buffer (Tris-HCl, SDS, and 20% glycerol) and detected using SDS-PAGE. The proteins were electrotransferred onto Immobilon-P PVDF membranes (0.45 µm, Millipore, USA) and probed with the appropriate primary antibodies against the following: p-Raf-1 (Ser 338) (sc-12358, Santa Cruz, 1∶200), caspase-8 p18 (G-1) (sc-166596, Santa Cruz, 1∶500), caspase-3 p17 (B-4) (sc-271028, Santa Cruz, 1∶500), JNK1 (D-6) (sc-137018, Santa Cruz, 1∶500), p-JNK1/2/3 (T183+Y185) pAb (BS4322, Bioworld Technology, 1∶500), ERK 1/2 (H-72) (sc-292838, Santa Cruz, 1∶200), p-ERK 1/2 (Thr202/Tyr204) (sc-16982, Santa Cruz, 1∶200), IκB-alpha (N-terminus) mAb (MB0106, Bioworld Technology, 1∶1000), β-actin (Sigma, 1∶5,000), and β-tubulin (Sigma, 1∶5000)). After incubation with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz, 1∶10,000) for 3-4 h, the membranes were washed with PBST. Immunoreactivity was visualized using an ECL system (Perkin Elmer, USA), and densitometry scanning of the intensity of the bands was quantified using ImageJ.

Western blotting was performed as previously described [29 (link),30 (link)]. In short, the cell lysates were denatured in a sample buffer containing sodium dodecyl sulfate (SDS), and equal amounts of total protein were separated on 8%–15% SDS-poly-acrylamide gels and transferred to nitrocellulose membranes. After blocking with 5% nonfat dry milk, the membranes were incubated overnight at 4 °C with the primary antibodies as indicated. The following antibodies were used: against active-caspase-3 (sc-271028), active-caspase-8 (sc-5263), active-caspase-9 (sc-133109), cleaved cleaved-poly adenosine diphosphoribose polymerase 1 (PARP-1) (sc-56196), cytochrome c (sc-13156), cytochrome c oxidase IV (COX IV) (sc-376731), Bcl-2 (sc-492), and Bax (sc-526) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-â-actin was purchased from Sigma Chemical Co. (St. Louis, MO, USA), and these antibodies against LC3, Atg5/12 conjugate, and Beclin-1 were purchased from Novus Biological Inc (Littleton, CO, USA). The following day, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies, and detection was performed using enhanced chemiluminescence (ECL) reagent (Amersham Life Science).

Total cell lysates were prepared and analyzed by Western blot as described previously [20 (link)]. We used the following primary antibodies—for the detection of cleaved caspase 3: monoclonal antibody (MAB10753) from MilliporeSigma, St. Louis, MO, USA) or monoclonal antibody (SC-271028) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and for PARP-1: SC-56196 from Santa Cruz (specific for the cleaved form) and #9542 from Cell Signaling Technology (Danvers, MA, USA) (recognizing full-length and cleaved PARP-1). Horseradish peroxidase-antibody conjugates (i.e., secondary antibodies) were obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). All antibodies were used according to the suppliers’ recommendations. For detection, SuperSignal West Pico PLUS Chemiluminescent Substrate was used (ThermoFisher Scientific, Waltham, MA, USA). Immunoblots were repeated to confirm the results. The uncropped Western blot images can be found at Figure S7.