Pab30596

Protein extracts (20 μg) prepared from the Kasumi-1 and Kasumi-6 cells were separated by 12% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). The membranes were blocked for 120 min in a skim milk blocking buffer, washed five times with Tris-buffered saline with 0.1% Tween 20 detergent (TBST) containing 0.1% Tween 20, and probed with primary antibodies against hexokinase 2 (HK2, 1:1000, PAB30271, Bioswamp), AKT (1:1000, PAB30596, Bioswamp), p-AKT (1:1000, ab38449, Abcam), mTOR (1:1000, PAB30674, Bioswamp), p-mTOR (1:1000, PAB36313-P, Bioswamp), Multidrug resistance-associated protein 1 (MRP1) (1:1000, ab23338, Abcam), P-glycoprotein (P-gp, 1:1000, 22,336-1-AP, Proteintech), and GAPDH (1:1000, PAB36269, Bioswamp) overnight at 4°C. The membranes were washed three times with TBST and incubated with secondary antibodies (goat anti-rabbit IgG, 1:20,000, PAB160011, Bioswamp) for 60 min [25 (link)]. Protein bands were examined using enhanced chemiluminescence (Tanon-5200, TANON) and analyzed using AlphaEase FC gel image analysis software.

Protein extracts (10 or 20 μg) prepared from LLCs were separated by 6% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore, Wisconsin, USA). The membranes were blocked with 5% milk in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 and incubated overnight at 4°C with specific primary antibodies against the following proteins: ALG-2-Interacting protein X (ALIX, 1:1000, PAB33277, Bioswamp, China), TSG101 (1:1000, PAB33278, Bioswamp), CD63 (1:1000, PAB33929, Bioswamp), beclin-1 (1:1000, PAB35215, Bioswamp), LC3I/II (1:1000, MAB37400, Bioswamp), p62 (1:1000, PAB35470, Bioswamp), AKT (1:1000, PAB30596, Bioswamp), p-AKT (1:1000, PAB43158-P, Bioswamp), mTOR (1:1000, PAB33332, Bioswamp), p-mTOR (1:1000, PAB36313-P, Bioswamp), and GAPDH (1:1000, PAB36269, Bioswamp). After three washes with PBS/Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit IgG (1:20000, SAB43714, Bioswamp) for 2 h at room temperature. Protein bands were visualized by enhanced chemiluminescence color detection (Tanon-5200, TANON, Shanghai, China) and analyzed using AlphaEase FC gel image analysis software.