Horseradish peroxidase hrp conjugated goat anti mouse igg

Western blot analyses were performed to validate the differentially expressed proteins. Every two samples in each group were mixed in one Western blot experiment, which was repeated for three times. Protein was extracted with lysis buffer and centrifuged to obtain the supernatant and determine the protein content by BCA assay. Each of the protein samples (30 μg) was subjected to SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA). The membranes were blocked for 1 h at room temperature with 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween 20 (TBST, Applygen Gene Technology Corp). Then they were probed overnight with primary anti-RALBP-1 (ab166655, Abcam, MA, USA, 1 : 1000) and anti-RBP-1 (ab154881, Abcam, MA, USA, 1 : 2000), followed by incubation with horse radish peroxidase (HRP) conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, 1 : 2500). Then they were developed with an enhanced chemiluminescence detection kit (Pierce Biotechnology, Inc., Rockford, IL). β-actin was used as a loading control.

The primary antibodies used were: mouse monoclonal anti-Rab2 (Abcam), rabbit polyclonal anti-Sar1 (Abcam), mouse monoclonal anti-GAPDH (Cell Signaling Technology), mouse monoclonal anti-His(Sigma), mouse monoclonal anti-Flag (Sigma), mouse monoclonal anti-Myc (Cell Signaling Technology), mouse monoclonal anti-HA (Cell Signaling Technology), mouse monoclonal anti-GroEL(Santa Cruz) and mouse monoclonal anti-Omp1 (Santa Cruz). The secondary antibodies used for Western blotting were: Horse Radish Peroxidase (HRP)-conjugated Goat Anti-Mouse IgG (Santa Cruz) and HRP-conjugated Goat Anti-Rabbit IgG (Cell Signaling) antibodies. Normal rabbit IgG was purchased from Santa Cruz. The secondary antibodies used for immunofluorescence were: goat anti-rabbit IgG (H + L) secondary antibody, Alexa Fluor^® 488 conjugate (Invitrogen), and donkey anti-rabbit IgG secondary antibody and Alexa Fluor^® 594 conjugate (Invitrogen).

Cytosolic extracts were prepared from Ramos cells treated with HCS with and without ZDEVD as well as bufalin (10 and 50 nM) for 24 h. Briefly, cells were washed in PBS and then resuspended in 50 μl of lysis buffer [20 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA (ethylenediaminetetraacetic acid), and 1 mM dithiothreitol (DTT)]. After sonication on ice for 3 min with a sonicator 3000 (Misonex Inc., Farmingdale, NY, USA), the protein concentrations were determined by the Bradford assay. Immunoblot assays were performed as per standard procedure. Briefly, equal amounts (50 μg) of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to PVDF membranes. Membranes were probed with the indicated antibodies. Secondary antibodies consisting of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (1:500 vol/vol) were purchased from Santa Cruz. Detection was performed by the enhanced chemiluminescence method from GE Healthcare (Little Chalfont, Buckinghamshire, UK).

Total proteins from breast cancer cells were lysed in modified RIPA lysis buffer (Beyotime, China) with freshly added protease inhibitors cocktail (Roche Diagnostics, Basel, Switzerland) and quantified by a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Then, 20 µg of total cellular extracts were separated by 10% SDS-PAGE and immobilized on polyvinylidene fluoride membrane (PVDF; EMD Millipore, Billerica, MA, USA). Following blocked by 5% skimmed milk (Sigma) for 2 h, the membrane was probed with primary antibodies against sirt1 and β-Actin (Abcam, Cambridge, MA, USA) overnight at 4 °C. Subsequently, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat-anti-mouse IgG (Santa Cruz biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. The protein bands were visualized using ECL detection reagent (Millipore, Billerica, MA, USA).

Antibodies used for protein analysis were as follows: monoclonal mouse anti-HA (1:5000; cat. no. H3663, Sigma-Aldrich, St. Louis, MO, USA), monoclonal mouse anti-Flag (1:5000; cat. no. F1804, Sigma-Aldrich), monoclonal rabbit anti-Alix-N-terminus (1:2000; cat. no. ab186429, Abcam, Cambridge, MA, USA), polyclonal rabbit anti-Vps4A/B (1:2000; cat. no. 17673-1-AP, Proteintech, Chicago, IL, USA), monoclonal mouse anti-Tsg101 (1:1000; cat. no. ab83, Abcam, Cambridge), Alexa Fluor-488-conjugated goat anti-mouse IgG (1:5,00; cat. no. A-11001, Invitrogen, Carlsbad, CA, USA), monoclonal mouse anti-GAPDH (1:5000; cat. no. sc-47724, Santa Cruz Biotechnology, Dallas, TX, USA), monoclonal mouse anti-Tubulin (1:5000; cat. no. sc-32293, Santa Cruz), polyclonal rabbit anti-Histone H3 (1:2000; cat. no. ab1791, Abcam), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000; cat. no. sc-2005, Santa Cruz), and HRP-conjugated goat anti-rabbit IgG (1:5000; cat. no. sc-2004, Santa Cruz).