Actin c 11

PBMCs or isolated CD4⁺ T cells were stimulated with Dynabeads Human T-Activator CD3/CD28 (Invitrogen, Life Technologies) (1 : 10 or 1 : 1 bead : cell ratio) for, respectively, 24 hours (RNA isolation/FACS staining) or 48 hours (protein analysis). Nonstimulated cells served as controls. For protein extraction, cells were harvested and washed with 1x PBS and extracted using cell lysis buffer (Cell Signaling, USA) according to the procedures provided by the manufacturer. A Bio-Rad assay was performed to determine protein concentrations of samples (Bio-Rad, Hercules, CA, USA). Protein lysates were separated via SDS-PAGE and then immunoblotted onto pure nitrocellulose membranes (Bio-Rad) and probed with specific antibodies: PI3 K p85 (Cell Signaling), PTEN (Cell Signaling), Phospho-PTEN (Ser380/Thr382/383) (Cell Signaling), and Actin (C-11) (Santa Cruz Biotechnology, USA). Secondary antibodies alexa fluor 680 donkey anti-rabbit IgG (Invitrogen) and donkey anti-goat IRDye 800CW (LI-COR Biosciences, Germany) were used. Proteins were detected using the Odyssey Imaging System (LI-COR Biosciences).

For immunoblotting, tumor protein was extracted from frozen tissue using PIERCE RIPA buffer, supplemented with additional 1X protease inhibitor cocktail containing 50mM NaF, 1mM Na₃VO₄, 17.5mM β-glycerophosphate, and 1mM PMSF (Calbiochem). For immunohistochemistry, tumor tissues fixed in 10% phosphate-buffered formalin were transferred to 70% ethanol after 48 hours to preserve antigens. Fixed tumor tissues were paraffin embedded and cut into 5μm sections for staining. Primary antibodies and dilution used for immunoblotting include: p53 (CM5, Novocastra) at 1:1000; p19ARF (ab80, Abcam) at 1:1000; p16 (M-156, Santa Cruz Biotech) at 1:1000; p15 (K-18, Santa Cruz Biotech) at 1:1000 and Actin (C-11, Santa Cruz Biotech) at 1:1000. HRP-conjugated Secondary antibodies (Jackson ImmunoResearch Laboratories) were used at 1:2000 dilution. An enhanced chemiluminescence (ECL) assay (PerkinElmer) was used and documented either on Kodak X-Omat Blue XB-1 film or by an AlphaEase digital imaging system (Alpha Innotech). Primary antibodies and dilution used for immunohistochemistry include: Ki-67 (Clone SP6, Lab Vision) at 1:200; cleaved caspase 3 (Biocare Medical) at 1:100; p53 (CM5, Novocastra) at 1:100.

The frozen myocardial tissues were lysed in radioimmunoprecipitation cell lysis solution (Beyotime Biotechnology, Shanghai, China), followed by high speed centrifugation (13,000 × g, 5 min) and β-cyanoalanine quantification. Cellular protein was separated by electrophoresis on SDS-PAGE gel and then transferred onto polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). After blocking, the blots were incubated with antibodies against GRP78 (N-20) (cat. no. sc-1050; 1/600; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), p-eIF2α (Ser49) (cat. no. sc-293100; 1/200; Santa Cruz Biotechnology Inc.), caspase-12 (A-14) (cat. no. sc-12395; 1/400; Santa Cruz Biotechnology Inc.) and GADD153 (F-168) (cat. no. sc-575; 1/500; Santa Cruz Biotechnology Inc.) at 4°C overnight. The eIF2α (FL-315) (cat. no. sc-11386; 1/500; Santa Cruz Biotechnology Inc.) and actin (C-11) (cat. no. sc-1615; 1/500; Santa Cruz Biotechnology Inc.) were used as loading controls. The appropriate horseradish peroxidase-conjugated secondary antibodies were added and the samples were incubated for 1 h at room temperature (1:6,000 dilution for p-eIF2a and caspase-12 and 1:10,000 dilution for GRP78, eIF2a, GADD153 and actin). The protein bands were detected with SuperSignal Ultra Chemiluminescent Substrate (Pierce, Rockford, IL, USA) on X-ray films (Koda).

An antibody recognizing H2AX (α-total H2AX, BL179) and 53BP1 (A300-272 A) was purchased from Bethyl Laboratories (Montgomery, TX). γ-H2AX phospho-specific antibody (JBW301) was obtained from Millipore (Billerica, MA). α-Artemis antibodies (E-18, K-14), and actin (C-11) were obtained from Santa Cruz Biotech (Santa Cruz, CA). α-Ku70 and α-Ku70/80 heterodimer specific antibodies (N3H10 and 162, respectively) were purchased from Genetex (Irvine, CA). K-H monoclonal antibody was produced at Case Western Reserve University. K-H polyclonal antibody (SAB1102247) was purchased from Sigma. S9.6, an antibody specific for Rloops (RNA:DNA hybrids) was provided by Dr Stephen H. Leppla (NIH, Bethesda, MD). PSF (A301-320A) and Xrn2 (A301-103A) are from Bethyl Laboratories (Montgomery, TX). RNAPII antibodies (sc-56767) is from Santa Cruz Biotech (Santa Cruz, CA).