Enhanced chemiluminescent reagent

After being pretreated with ISE, RAW264.7 macrophages were stimulated with LPS or L1CM for 30 min, and then harvested in RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 1 × protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN), and 1 × phosphatase inhibitor cocktail (Sigma-Aldrich). Proteins were resolved in 7.5% acrylamide gels and then transferred to nitrocellulose membranes. The membrane was blocked with 5% non-fat milk in Tris-buffered saline before being incubated, respectively with specific primary antibodies for the following proteins: IκB-α (1:1000), phosphor-p44/p42 (Thr202/Tyr204) (p-ERK) (1:1000), phosphor-p38 (Thr202/Tyr204) (p-p38) (1:1000), and Phospho-SAPK/JNK (Thr183/Tyr185) (p-JNK) (1:1000), phosphor-STAT3 (p-STAT3) (1:1000), ERK (1:1000), JNK (1:1000), p38 (1:1000), STAT3 (1:1000) (all from Cell Signaling Technologies, Danvers, MA), and β-actin (1:5000, Sigma-Aldrich). The membranes were next incubated with horseradish peroxides (HRP)-conjugated secondary antibodies followed by exposure to enhanced chemiluminescent reagents (Millipore, Burlington, MA).

Total protein was extracted using radioimmuno-precipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China). Then, a bicinchoninic acid (BCA) kit was used to quantify protein concentration and separated by gel electrophoresis (Beyotime). The loading volume was 20 μg. Following this, protein samples were transferred onto membranes. Membranes were sealed with bovine serum albumin (BSA) (Beyotime) for 1 h and incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibody (1:2000, ab9485, Abcam) for 1 h. Protein bands were subsequently detected using enhanced chemiluminescent reagents (Millipore). Protein expression was quantified using ImageJ software, with GAPDH serving as the loading control. The primary antibodies used were as follows: anti-PI3K (human and mouse; dilution: 1:1000; catalogue number: ab151549, Abcam, UK), anti-p-PI3K (human and mouse; dilution: 1:1000; catalogue number: ab182651, Abcam), anti-AKT (human and mouse; dilution: 1:500; catalogue number: ab8805, Abcam), anti-p-AKT (human and mouse; dilution: 1:500; catalogue number: ab38449, Abcam), and anti-GAPDH (human and mouse; dilution: 1:3000; catalogue number: AF7021, Affinity, USA).

Cells were lysed, and proteins were extracted and quantified. The cell lysates were incubated with specific antibodies or the anti-IgG control for 3–4 h at 4 °C with rotation. Then, samples were incubated with protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C with rotation. The beads were pelleted using centrifugation at 1000× g for 30 s at 4 °C; then, they were boiled for 10 min at 95 °C and resolved with 10% SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to PVDF membranes (Millipore, Bedford, MA, USA), and the membranes were blocked in 5% skim milk and incubated overnight at 4 °C with anti-AHNAK or anti-IGF-1R at a dilution of 1:200. After washing with TBST, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the membranes were incubated with enhanced chemiluminescent reagents (Millipore) and exposed to autoradiography film in the dark.

CHO culture supernatant (1 mL) was incubated with rabbit anti-human α-chain-specific antibody (30 μL, Sigma-Aldrich) overnight at 4°C. Protein-A sepharose (50 μL, Sigma-Aldrich) equilibrated in PBS was added and the mixture incubated for 4 h at 4°C then centrifuged (3000 ×g, 4°C, 1 min). The resulting pellet was washed three times with 1 mL of PBS. The immunoprecipitated protein was separated by reduced or nonreduced SDS-PAGE, and then transferred to polyvinylidene difluoride membranes. Membranes were blocked overnight at 4°C by incubating with 10% skim milk in PBST, then incubated for 2 h at 37°C with the following primary antibodies, respectively: mouse anti-human IgA, α-chain specific (1 : 3000; Abcam, Cambridge, MA, USA); mouse anti-human, κ-chain specific (1 : 3000; Sigma Aldrich); mouse anti-human, J chain specific (1 : 2500; Abcam); and mouse anti-human, SC specific (1 : 3000; Sigma Aldrich). Bound antibodies were detected with an HRP-conjugated goat anti-mouse IgG (1 : 3000; Sigma Aldrich) in combination with enhanced chemiluminescent reagents (Millipore, Bedford, MA, USA).

The cellular proteins were extracted from 10⁷ cultured cells after drug treatments using RIPA lysis buffer (Promega, USA). Western blotting analysis was performed to determine cellular responses targeting apoptosis-related proteins including Bcl-2 and Bax. Briefly, C6 and U-87MG cells were treated with different doses of DMAMCL for 24 hr at 37°C. Then cells were washed twice with cold phosphate buffered saline (PBS) and lysed in RIPA buffer containing 1mmol/l PMSF (Pheylmethylsulfonyl fluoride, Sigma, USA) on ice for 30 min. The total proteins of cells were obtained by centrifugation at 12000 rpm for 15 min. Next, the lysate proteins were resolved by SDS-PAGE and transferred to the Immobilon-P membranes (Millipore, Billerica, MA, USA). The membranes were blocked by 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and immunoblotted with the antibodies against β-actin, Bcl-2 and Bax (Cellsignaling Technology, USA) at 4°C overnight. Then membranes were incubated with HRP-conjugated secondary antibody for 1.5 hr at room temperature. The targeting antibodies were stained with enhanced chemiluminescent reagents (Millipore, Billerica, MA, USA) and visualized by ChemiScope 3600 Mini systerm (Clinx Science Instruments, China).