Goat anti mouse igg

Yeast total extracts were prepared as previously described (Knop et al., 1999 (link)). 1.5 × 10⁸ cells from OD₆₀₀ = 0.4–0.6 cultures were resuspended in 1150 µl lysis buffer (0.24 M NaOH, 1% β-mercaptoethanol, 1 mM EDTA, 1 mM PMSF, 5 µM Pepstatin A, 10 µM Leupeptin). After incubation on ice for 20 min, 150 µl 55% trichloroacetic acid (TCA) was added to precipitate proteins on ice for 20 min. The mixture was centrifuged at 16100 rpm at 4°C for 10 min. The pellet was resuspened in 250 µl HU buffer (8 M urea, 5% SDS, 200 mM Tris-HCl [pH 6.8], 1 mM EDTA, 5% β-mercaptoethanol, and 1% bromophenol blue) and incubated at 65°C for 10 min, followed by 16100 rpm centrifugation at RT for 5 min. The supernatant was used for subsequent analyses. Immunoblotting was performed with primary antibodies: rabbit-anti-GFP (1:2000) (Abcam), and mouse-anti-PGK1 (1:1000) (Abcam). Mouse-anti-rabbit-IgG (1:10,000) (Santa Cruz) and goat-anti-mouse-IgG (1:10,000) (Santa Cruz) were used as secondary antibodies.

Relative protein expression of five apoptosis-related genes (Bcl-2, p53, Bax, Cyt c, and Caspase-3) was measured using Western blot assay followed the method described in previous study (28). The first antibody (1:100), secondary antibody (1:1000), monoclonal β-actin antibody (1:1000), and goat antimouse IgG (1:1000) of Bcl-2, p53, Bax, Cyt c, and Caspase-3 were purchased from Santa Cruz Biotechnology, USA. The signal was detected using X-ray films (TianGen Biotech Co. Ltd., Beijing, China). The optical density of each band was measured using Image VCD gel imaging system (Beijing Sage Creation Science And Technology Co. Ltd., China).

A rabbit anti-human Kir2.1 polyclonal antibody was purchased from Alomone labs (Jerusalem, Israel), andpolyclonal anti-MRP1/ABCC1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibodies were purchased from Santa Cruz Inc. (CA, USA). Horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) and goat anti-mouse IgG were obtained from Santa Cruz Inc. U0126, the MEK inhibitor, and staurosporine, the PKC inhibitor, were purchased from Selleck Chemicals (Houston, TX, USA). All three chemotherapeutic drugs, Cisplatin (DDP; Shandong, China), Etoposide (VP-16; Jiangsu, China) and Adriamycin (ADM; Jiangsu, China), were obtained from commercial sources and were dissolved according to the manufacturer’s instructions.

Retinal samples or samples of cells were sonicated in lysis buffer, namely mammalian protein extraction reagent (MPER; HyCell). Identical quantities of denatured proteins (40 μg/30 μl/well) then underwent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad) as described previously [10 (link)]. After separation, the protein bands were transferred to a polyvinylidene difluoride membrane, which was treated for 12 h at 4 °C with the following primary antibodies, mouse monoclonal anti-β-actin antibody (AC-15; 1:2000; ab6276)/anti-HIF-1α antibody (1:200; H1alpha67-ChIP Grade; Abcam Inc.), rabbit polyclonal anti-VEGF antibody (A-20; 1:200; sc-152)/anti-PKM2 antibody (1:500; ab38237), or rabbit monoclonal anti-RBP2 antibody (ab177486; 1:1000; Abcam Inc.). The blots were next treated with relevant secondary antibody, HRP-conjugated goat anti-rabbit IgG (1:5000; Santa Cruz Biotechnology Inc.) or goat anti-mouse IgG (1:5000; sc-2005) at 37 °C for 1 h. Dilution of primary/secondary antibodies was carried out in 5% fat-free skimmed milk. Finally, the membranes were processed using an enhanced chemiluminescent analysis system (HyCell) and exposed to an X-ray film (Fujifilm). The amount of each protein was then evaluated by scanning densitometry.

The MDSCs were fixed with an ice-cold 4% paraformaldehyde, incubated with 3% H2O2 for 30 min., and blocked with goat serum. The cells were then incubated with neuron-specific enolase (NSE) (1 : 100, sc-51880) overnight at 4°C, followed by a goat anti-mouse IgG (1 : 100; sc-2005, Santa Cruz Biotechnology, USA). Next, cells were incubated with DAB solution (D3939, Sigma, USA).

Western blot analysis was performed as described previously (12 (link)) using the following antibodies: mouse anti-AKT (BD Biosciences), rabbit anti-pAKT, rabbit anti-p4E-BP1, rabbit anti-4E-BP1, rabbit anti-pS6, mouse anti-S6, rabbit anti-pERK, rabbit anti-ERK, rabbit anti-pan-RAS (Cell Signaling, Beverly, MA, USA). Mouse anti-GAPDH (HyTest, Turku, Finland) or mouse anti-β-Actin (Sigma) were used as loading controls. Goat anti-mouse IgG, donkey anti-goat IgG, goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology Inc.), and Goat anti-mouse IgG1 or Goat anti-mouse IgG2b (Southern Biotech, Birmingham, AL, USA) conjugated to horseradish peroxidase were used as secondary antibodies. Enhanced chemiluminescence was used for detection (Amersham Bioscience, Freiburg, Germany). Also, donkey anti-mouse IgG or donkey anti-rabbit (LI-COR Biotechnology, Bad Homburg, Germany) labeled with IRDye infrared dyes were used for detection. Representative blots of at least two independent experiments are shown.

Cell lysates were prepared using RIPA buffer containing a protease and phosphatase inhibitor cocktail (Calbiochem, La Jolla, CA, USA). The total protein was quantified using Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (20 μg per lane) from the samples were separated by 8% or 10% SDS-PAGE and probed with the indicated antibodies. Antibodies against survivin (#2808), ERK (#9102), pERK (#9106), JNK (#9252), pJNK (#9251), p38 (#9212), and pp38 (#9216) were purchased from Cell Signaling Technology (Danvers, MA, USA), and antibodies against BCl (#sc-783), Bax (#sc-493), caspase-3 (#sc-7148), caspase-9 (#sc-7885), PARP (#sc-7150), β-actin (#sc-47778) and GAPDH (#sc-32233) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The secondary horseradish peroxidase-conjugated antibodies used were goat anti‑rabbit IgG and goat anti‑mouse IgG (SC‑2004 and SC-2005, respectively; Santa Cruz Biotechnology, Dallas, TX, USA). Relative intensities of the bands were analyzed with a GS‑710 Image Densitometer (Bio‑Rad Laboratories). Results are representative of at least five independent experiments.