Goat anti rabbit igg secondary

Cells (5 × 10⁵) were collected at various time points and then washed with ice-cold PBS twice before adding lysis buffer (M-PER Mammalian Protein Extraction reagent, Thermo Scientific, Cat No. 78501) and cocktail inhibitor (Sigma, 5 μg/ml, Cat No. P8340). Equal amounts of protein were loaded into each well and separated by NuPAGE 4–12% Bis-Tris gel, followed by transfer onto PVDF-membranes (Bio-Rad, Cat No. 162-0177). Membranes were blocked with 5% non-fat milk in PBS-T for 1 h at room temperature. The blots were then incubated with antibodies against GRPR, Caspase-3, p-AKT1, AKT1, p-AKT2, AKT2, p-mTOR, mTOR, p-MAPK, MAPK, p-RPS6, RPS6, p-p38 MAPK, p38 MAPK CD133, Sox2, and ALDH for 1 h at 4°C. Goat anti-rabbit IgG secondary (1:5000; Santa Cruz Biotechnology, Cat No. sc-2004) was then incubated for 45 min at room temperature. Immunoblots were developed by using the chemiluminescence detection system (PerkinElmer, Cat No. NEL105) and autoradiography was performed. β-actin was used as a loading control.

Cells were washed twice with ice-cold PBS before the addition of lysis buffer (m-PER Mammalian Protein Extraction reagent, Thermo Scientific, Waltham, MA) and inhibitor cocktail (Sigma, 5μg/mL). Protein concentration was quantified by the Bio-Rad method. 35μg of protein were loaded into each well of a 12.5% SDS-PAGE gel, followed by transfer onto PVDF-membranes (Bio-Rad, Hercules, CA). Membranes were blocked with 5% non-fat dry milk in PBS-T for 1 hour at room temperature. The blots were then incubated with antibodies against the following human proteins: p-mTOR (1:1000; Cell Signaling Technology, Danvers, MA); mTOR (1:1000; Cell Signaling Technology); p-Akt (1:1000; Cell Signaling Technology, Danvers, MA); Akt (1:1000; Cell Signaling Technology); p-S6 ribosomal protein (1:1000; Cell Signaling Technology); S6 ribosomal protein (1:1000; Cell Signaling Technology); caspase-3 (1:1000; Cell Signaling Technology); and actin (1:1000; Cell Signaling Technology) for 1 hour at 4°C. Blots were rinsed 3×10min, then incubated with goat anti-rabbit IgG secondary (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA) for 45 minutes at room temperature. Immunoblots were developed by using the chemiluminescence detection system (PerkinElmer, Waltham, MA), according to the manufacture's protocol, and autoradiography.

Cells (2 × 10⁵/mL) were seeded into 6-well plates and treated with different BQ concentrations (0, 10, and 20 μmol/L) for 24 h. The cells were washed twice with PBS and then lysed in RIPA lysis buffer (1 mM PMSF; Beyotime Biotechnology, Nantong, China) on ice for 10 min, sonicated for 5 s, and centrifuged at 12000 ×g for 10 min at 4°C. Protein concentration in the supernatant was assayed by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA). The protein was mixed with 6∗SDS sample buffer and boiled for 5 min. Equal amounts of protein were electrophoresed on 10% SDS-PAGE and then electroblotted onto polyvinylidene fluoride membranes. The membrane was incubated overnight at 4°C with anti-G6PD polyclonal antibodies (RbpAb to G6PD; Abcam, Cambridge, UK) and anti-β-actin polyclonal antibodies (mouse monoclonal IgG; Santa Cruz, CA, USA). Secondary goat anti-rabbit IgG or goat anti-mouse IgG (Santa Cruz, CA, USA) was used according to the species of primary antibody. Chemiluminescence was measured by a chemiluminescent imaging system (Tanon-5200, Shanghai, China) using Immobilon™ Western Chemiluminescent HRP Substrate (Millipore Corporation, Billerica, USA).