Anti gapdh mab 60 004 1 ig

The synthetic miRNA probe miR-4634 (#YD00611033) (QIAGEN, Germany), was resuspended in RNase-free water at 25 µM, probe sequence 5′-CGGGCCGGTCGCGC-3′. RAD001 (Selleckchem, Houston, TX, USA). The primary antibody anti-GAPDH mAb (60,004–1-Ig) at a 1:50,000 dilutions was purchased from Proteintech Group. Other primary antibodies were purchased from Cell Signaling Technology, such as cleaved-PARP (#5625) at a 1:1,000 dilutions, DR4 (#42,533) at a 1:1,000 dilutions, DR5 (#8,074) at a 1:1,000 dilutions, c-Myc (#18583) at a 1:1,000 dilutions, Bcl-xL (#2764) at a 1:1,000 dilutions, Bak (#12105) at a 1:1,000 dilutions, Bax (#5023) at a 1:1,000 dilutions, Bad (#9239) at a 1:1,000 dilutions.

Cells were lysed in mammalian cell lysis buffer (MCLB) containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% NP-40, and supplemented with EDTA-free protease inhibitor cocktail (04693159001, Roche) and 1 mM phenylmethyl sulfonyl fluoride (PMSF, 0754, Amresco). Protein concentrations were measured using the Bradford assay (5000201, Bio-Rad). Then, the protein was boiled in 5×sodium dodecyl sulfate sample buffer. The protein from each sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane (Millipore). The membranes were blocked and incubated with primary antibodies (diluted 1:1000) in primary antibody dilution buffer (Beyotime Biotechnology). The primary antibodies used were anti-HA-Tag rabbit mAb (C29F4, CST), anti-GAPDH mAb (60004-1-Ig, Proteintech), and anti-PD-L1 mAb (ab205921, Abcam). The membranes were then incubated with goat anti-rabbit IgG (H+L) secondary antibody (31460, ThermoFisher) or goat anti-mouse IgG (H+L) secondary antibody (31430, ThermoFisher). Images were acquired using the ChampChemi imaging system (Sage Creation Science).