Anti bax sc 493

Western blot assays were performed on rat lungs stored at -80°C. Samples were lysed in buffer containing phosphatases and proteases inhibitors. Total proteins (100 μg) were separated on 4–12% NuPage Bis-Tris gels (Life Technologies, Carlsbad, USA) and transferred onto PVDF membranes (GE Healthcare, Freiburg, Germany). The membranes were incubated over-night at 4°C with a primary antibody: mouse monoclonal anti-p53 (clone PAb122, LS-C63152, LSBio, Seattle, USA), rabbit polyclonal anti-p21 (sc-397, Santa Cruz, Le Perray en Yvelines, France), rabbit polyclonal anti-Bax (sc-493, Santa Cruz), mouse monoclonal anti-MDM2 (MCA1709, AbDserotec) or mouse monoclonal anti-βactin (clone AC-74, A5316, Sigma-Aldrich). Incubation with a secondary HRP antibodies (anti-mouse sc-2314 or anti-rabbit sc-2313, Santa Cruz) was performed for 1 hour at room temperature. HRP signals were detected by ECL substrate (Immun-Star WesternC Kit, Biorad, Marnes-la-Coquette, France) using a ChemiDoc XRS+ (Biorad) and blots were quantified and normalized by actin signal with ImageLab software (Biorad).

The following reagents were used in this study. Sorafenib, oxaliplatin, and paclitaxel were purchased from Selleck (Selleck, China). Cisplatin, irinotecan, and 5-fluorouracil (5-Fu) were purchased from Sigma (Sigma-Aldrich, USA). Targeted specific siAFPs were synthesized by Genepharma (Shanghai, China), and the sequences are listed in Supplementary Table 1. SiFas (sc-29311) was purchased from Santa Cruz Biotechnology. For Western blot analysis, the following antibodies were used. Anti-PARP (#9532), anti-caspase 3 (#9662), anti-caspase 9 (#9502), anti-myc-tag (#2278), anti-Cyto c (#11940), anti-β-actin (#4970 S) and anti-GAPDH (#2118) antibodies were purchased from Cell Signaling Technology. Anti-AFP (SC-166325), anti-Fas (SC-1204 and SC-8009), anti-FasL (SC-834), anti-FADD (SC-271748), anti-Bcl2 (SC-509), anti-Lamin A/C (sc-7292), and anti-Bax (SC-493) antibodies were purchased from Santa Cruz Biotechnology. An anti-caspase 8 antibody (#AF1650) was purchased from R&D Systems, anti- IR-β (ab69508) antibodies were purchased from Abcam, and an anti-HuR antibody (#07-468) was purchased from Millipore.

HaCaT cells were lysed with RIPA lysis buffer (WSE-7420, Atto, Tokyo). Proteins were then extracted by centrifuging at 8,000xg for 15 min at 4°C and collecting supernatants. Cell nuclear and cytoplasmic extracts were prepared using NE-PER extraction reagents (#78833, Thermo Fisher Scientific, Waltham, IL, USA) according to the manufacturer's protocol. Next, 25–50 µg of proteins was resolved by 10% SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Carrigtwohill, Ireland), which were subsequently blocked with 5% skim milk in 1X PBS for 2 h at room temperature, incubated with primary antibodies and then horseradish peroxidase- (HRP-) conjugated anti-IgG antibody. Anti-Nrf2 (ab137550), anti-HO-1 (ab13243), anti-NQO-1 (ab34173), antioccludin (ab216327), and anticlaudin (ab15098) were purchased from Abcam (Cambridge, MA, USA), anti-PARP (#9542S) from Cell Signaling Technology (Danvers, MA, USA), anti-Bax (sc-493) from Santa Cruz Biotech (Paso Robles, CA, USA), anti-Bcl-2 (OP60T) from Oncogene Research Products (La Jolla, CA, USA), and anti-β-actin (A1978) from Sigma-Aldrich (St. Louis, MO, USA). Blots were detected by enhanced chemiluminescence (BioRad, Hercules, CA, USA) and protein band intensities were quantified using GelPro V3.1 software (Media Cybernetics, Rockville, MD, USA).

Our previous experiments that showed that Bcl-2 (B-cell lymphoma 2) is directly targeted by miR-130a-3p and miR-148a-3p. They also showed that expression of XIAP (X-linked inhibitor of apoptosis protein) is downregulated by miR-130a-3p, and that expression of Bim (Bcl-2-like protein 11) is downregulated by miR-148a-3p^{13 (link)}. Therefore, we analysed the effect of miR-148a-3p expression on expression of these targets in combination with further downstream targets in the p53-dependent apoptosis-pathway (Bax and Caspase-9). For miR-130a-3p, we analysed Bcl-2 and XIAP and again Caspase-9 in the p53-dependent apoptosis-pathway. Cells were lysed 48 h after transfection with either mimic or inhibitor of miR-130a-3p or miR-148a-3p and processed according to a standard protocol as described previously^{58 (link)}. Primary antibodies were purchased from BD Bioscience (anti-Bcl-2 #551097, anti-Bim #559685, anti-XIAP #610716), Santa Cruz Biotechnology (anti-Bax #sc-493), and Cell signaling (anti-Casp9 #9502). Three independent Western experiments were performed.

Bax and Bcl-xl protein levels were measured by Western blotting. Briefly, myocardial tissue protein extracts were prepared from control and treated samples in different groups, and suspended in PBS containing protease inhibitor cocktail. 30μg of protein was loaded onto 10% FastCast Acrylamide gel (Bio-Rad Laboratories Ltd). SDS-PAGE electrophoresis, immunoblotting, and protein detection were done for Bax, Bcl-xl and ß-actin using anti Bax (sc-493, Santa Cruz), anti Bcl-xl (sc-8392, Santa Cruz) and anti ß-actin (sc-47778, Santa Cruz) antibodies. Band intensity was analyzed by ChemiDoc^™ imaging system with Image Lab^™ software version 5.1 (Bio-Rad Laboratories Inc., USA). The results were normalized with ß-actin.

Cells were lysed with radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China). After the protein concentration was determined with BCA protein assay kit (Thermo Fisher Scientific), equal protein aliquots (25 μg per lane) were resolved on SDS gel electrophoresis and Western blotting assay was performed with standard protocol. The expression of target proteins was detected by using enhanced chemiluminescence system (Bio-Rad, Richmond, CA, USA) and quantified by densitometry with Image J software (http://rsb.info.nih.gov/ij/, Bethesda, MD, USA). Protein expression was normalized by the loading control (GAPDH) expression. Antibodies against FAM46C (ab169699), CDK1 (ab32384), XIAP (ab2541) and Ras (ab52939) were purchased from Abcam (Cambridge, MA, USA). Anti-Bcl-2 (sc-492) and anti-Bax (sc-493) were from Santa Cruz Biotech. (Santa Cruz, CA, USA). Antibodies against Cyclin B1 (#4135), PCNA (#13110), p-MEK1/2 (#8211), MEK1/2 (#9122), p-ERK1/2 (#4376), ERK (#4695) and GAPDH (#5174) were purchased from Cell Signaling (Danvers, MA, USA).