Anti bcl 2 sc 7382

LysoPC (#L1381), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, #M2128), nifedipine (#7634), KB-R7943 (#K4144), LaCl3 (#449830), ruthenium red (#R2751), RN1734 (#0658), SKF96365 (#S7809) and Pyr3 (#P0032) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies anti-TRPC1 (sc-133076), anti-TRPC3 (sc-514670), anti-TRPC4 (sc-15063), anti-TRPC5 (sc-18737), anti-Bax (sc-6236), and anti-Bcl-2 (sc-7382) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-TRPC6 (BA3394), anti-TRPC7 (PB0272) were purchased from Boster Biotechnology (Wuhan, China). Anti-caspase-3 (#9662), anti-Akt (#9272) and anti-pAkt(S473) (#4060) were from Cell Signaling Technology (Danvers, MA, USA). S-MEM without Ca²⁺ medium (#11380037) were obtained from Gibco. Other reagents were described as in specified.

Cells were plated in six-well plates. After treatment with ISL-17 (20 or 40 μM) and ISL (40 μM) for the indicated time, the cells were scraped from the plate. The collected protein samples were then electrophoresed on 10% SDS/PAGE gels, and electroblotted onto a PVDF membrane. At room temperature, the membranes were blocked with 5% nonfat milk for 2 h. Primary antibodies were incubated with membranes at 4°C overnight. Then, the membranes were washed with TBST for three times and incubated with secondary antibody at room temperature for 1 h. The immune-reactive complexes were analyzed with ECL kit (Bio-Rad, Hercules, CA, U.S.A.). The anti-Bcl-2 (sc-7382, 1:1000) and anti-Bax antibody (sc-20067, 1:1000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Antibodies including anti-Cyclin B1 (#4135, 1:2000), anti-Cdc2 (#9116, 1:1000), anti-Cleaved PARP (#5625, 1:1000), anti-p62 (#8025, 1:1000), anti-LC3B (#3868, 1:1000), anti-Beclin 1 (#3495, 1:1000), anti-AKT (#4691, 1:1000), anti-phospho-AKT (#4060, 1:2000), anti-mTOR (#2983, 1:1000), anti-phospho-mTOR (#5536, 1:1000), anti-rabbit IgG-HRP (#7074, 1:1000) and anti-mouse IgG-HRP (#7076, 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.).

Human gastric cancer cell lines SGC-7901 and BGC-823 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were routinely cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C in a humidified incubator with an atmosphere of 5% CO₂. N-acetyl-L-cysteine (NAC), L-glutathione (GSH) and cisplatin were purchased from Sigma (St. Louis, MO, USA). BMS-582949 and SP600125 were purchased from Selleck Chemicals (Houston, TX, USA). WZ35 was synthesized in our laboratory as previously described [13 (link)]. Antibodies including anti-Bcl-2 (sc-7382, 1:50), anti-TrxR1 (sc-28,321, 1:200), anti-GAPDH (sc-47,724, 1:200), mouse anti-rabbit IgG-HRP (sc-2357, 1:2000) and m-IgGκ BP-HRP (sc-516,102, 1:2000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies including anti-p-p38 (4631, 1:1000), anti-p38 (9212, 1:1000), anti-p-JNK (4668, 1:1000) and anti-JNK (9252, 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-Ki-67 (ab15580, 1:1000) antibody was purchased from Abcam (Cambridge, MA, USA). FITC Annexin V apoptosis Detection Kit I and Propidium Iodide (PI) were purchased from BD Pharmingen (Franklin Lakes, NJ, USA).

~ 1 × 10⁶ cells were seeded into a T-75 flask overnight. The cells were administered with 1 mL of fresh medium containing 10 µM Elli, either free or encapsulated in naked or hNET-homing nanovehicles. After 24 h, cells were lysed with RIPA buffer. 10 µg of the total proteins were loaded in loading buffer (50 mM Tris/HCl, 2% SDS and 20% glycerol), separated and electrotransferred onto a PVDF membrane. After blocking of nonspecific binding with 10% dried skimmed milk, anti-Bcl-2 (sc-7382, Santa Cruz Biotechnology, 1:200), anti-p53 DO-1 (sc-126, Santa Cruz Biotechnology, 1:250), anti-survivin (ab76424, Abcam, 1:5000) and anti-GAPDH G-9 (sc-365062, Santa Cruz Biotechnology, 1:700) antibodies were incubated with the membranes for 16 h at 4 °C. After washing, HRP-labeled anti-rabbit antibody (SAB3700831, Sigma-Aldrich) for survivin or anti-mouse antibody (P0260, Dako) for Bcl-2, p53 and GAPDH, both 1:5000, were used. Chemiluminiscent substrate Clarity Western ECL Blotting Substrate (Bio-Rad) was used, followed by detection on Azure c600 imager (Azure Biosystems). The densitometric analyses were performed using Fiji ImageJ (National Institute of Health).

Cells were lysed with IP lysis buffer containing a 10% cocktail (B14001, Bimake, Houston, TX, USA), and the protein concentration was determined using a BCA kit (Pierce Chemical, Rockford, IL, USA). SDS-PAGE was performed using 30–50 μg of total cell protein. Then, the protein was transferred to a PVDF membrane, blocked with 5% skim milk at room temperature, and incubated with the indicated antibodies. The primary antibodies used were anti-LMP1 (M0897, 1:500) purchased from DAKO (Glostrup, Denmark), and anti-LC3 obtained from NOVUS (NB100-2220, 1:500, Littleton, CO, USA). Anti-BNIP3 (ab10433, 1:1000) and anti-Beclin1 (ab207612, 1:1000) were purchased from Abcam (Cambridge, MA, USA). Anti-p-Akt (ser473, 9270, 1:1000), anti-p-ERK1/2 (4370, 1:1000), anti-p-JNK (Thr183/Tyr185, 9251, 1:1000), anti-p-JAK3 (Tyr980/981, 5031, 1:1000), anti-rabbit IgG-HRP (14708, 1:2000), and anti-mouse IgG-HRP (14709, 1:2000) antibodies were obtained from cell signaling technology (Danvers, MA, USA). Anti-Bcl-2 (sc-7382, 1:500), anti-p-P38 (thr180/try182, sc-4511, 1:1000), anti-HIF1α (sc-53546, 1:500), and anti-β-actin (sc-8432, 1:2000) antibodies were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). Blots were analyzed using a chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA).

MG132 and cycloheximide (CHX) were purchased from Selleck Chemicals (Houston, TX, USA). All antibodies used in this study are listed below: Mouse monoclonal Anti‐β‐actin (A1978, Sigma‐Aldrich, St. Louis, MO, USA), anti‐AKR1C3 (A6229, Sigma‐Aldrich), anti‐AR‐V7 (AG10008, Precision Antibody, Columbia, MD, USA), anti‐ubiquitin (sc‐47721, Santa Cruz, Santa Cruz, CA, USA), anti‐Bcl‐2 (sc‐7382, Santa Cruz); rabbit polyclonal anti‐AR (N‐20) (sc‐816, Santa Cruz) and anti‐B4GALT1 (A8546, ABclonal, Woburn, MA, USA).

Streptozotocin (STZ) was purchased from Merck & Co. Inc. (Regierungsbezirk Darmstadt, Hesse-Damstadt, Germany). The amplification primers were provided by GenScript Biotechnology Co. Ltd. (Nanjing, China). RNA extraction kits, reverse transcription kits, and amplification kits were obtained from Yeasen Biotech Co. Ltd. (Shanghai, China). Superoxide dismutase (SOD), malondialdehyde (MDA), and the radioimmunoassay kits for insulin were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Bicinchoninic acid (BCA) protein assay kits, RIPA lysis buffer, protease inhibitor cocktail, and a kit for sodium dodecyl sulfate-polyacrylamide gel electrophoresis were obtained from Wuhan Servicebio Technology Co. Ltd. (Wuhan, China). Hematoxylin-eosin was purchased from Wuhan Servicebio Technology Co. Ltd. (Wuhan, China). Rabbit anti-p47^phox(p47) (sc-17845), anti-(Bcl-2) (sc-7382), and anti- (vWF) (sc-365712) were purchased from Santa Cruz Biotechnology. Rat anti-α-SMA (19245S), anti-Bax (2772S), anti-eNOS (4231S), anti-rabbit IgG (5151P), anti-rat IgG (5257P), and horseradish peroxidase-linked antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).