Ac033

Cells were lysed in RIPA Lysis Buffer (Beyotime) and mixed with PMSF (Beyotime), and protein concentrations of the resulting lysates were quantified using a BCA Protein Assay Kit (Beyotime). A total of 30 μg of protein from each sample was separated using SDS-PAGE on a 10% gel and transferred to a polyvinylidene fluoride membrane. The membranes were probed with the following antibodies: 1:1000 rabbit polyclonal anti-CHERP antibody (ab15951, Abcam); 1:1000 rabbit polyclonal anti-cleaved caspase-3 antibody (AC033, Beyotime); mTOR substrate antibodies (9862, CST); 1:800 polyclonal anti-DR5/TNFRSF10B antibody (BS60081, Bioworld); 1:1000 polyclonal anti-Bip antibody (3177, CST); 1:1000 CHOP polyclonal antibody (2895, CST); 1:500 AKT polyclonal antibody (4691, CST); 1:1000 polyclonal anti-P-Akt (Ser473) antibody (4060, CST); 1:800 polyclonal anti-cleaved caspase-8 antibody (E1A5267, EnoGene); and 1:800 polyclonal anti-ATF4 antibody (E2A6008, EnoGene). For the cell cycle protein assay, either a cell cycle regulation antibody sampler kit (9932, CST) or a 1:2000 α-tubulin mouse polyclonal antibody (AT819, Beyotime) was used. The secondary antibodies utilized were horseradish peroxidase-conjugated goat anti-mouse (A0216, Beyotime) and goat anti-rabbit IgG (A0208, Beyotime).

RIPA Lysis Buffer (Beyotime) was used to extract total protein, and BCA Protein Assay Kit (Beyotime) was used to quantify the protein. Afterwards, protein samples (30 µg) were separated by 10% SDS-PAGE gel and transferred to PVDF membranes (Beyotime). Next, the membranes were blocked with skimmed milk for 2 h. After incubating with primary antibodies against Bcl-2 (26Kda, 1:2,000, BA0412, Boster, Wuhan, China), Bax (20Kda, 1:1,500, BA0315-2, Boster), Cleaved-caspase 3 (17Kda, 1:1,000, AC033, Beyotime), FOXO1 (82Kda, 1:2,000, AF603, Beyotime), or GAPDH (36Kda, 1:2,000, A00227, Boster), the membrane was then incubated with HRP Conjugated AffiniPure Goat Anti-rabbit/mouse IgG (H + L) (1:10,000, BA1056, Boster). The protein signals were visualized using BeyoECL Star (Beyotime). EasySee Western Marker (25-90Kda, DM201-01, Transgen Biotech, Beijing, China) was used as a molecular weight standard.

After the required treatments, protein extracts were prepared as follows. Briefly, cells were washed with cold PBS and lysed in RIPA buffer containing PMSF (Wuhan Goodbio, Lot: 170118). Equal amounts of the protein samples were resolved by 12% SDS-PAGE and transferred to an Immobilon-P membrane (Biosharp). Each membrane was, respectively, probed with a polyclonal antibody against TRAIL protein (Abcam ab2435, 1:1,000), human DR4 (Flarebio CSB-PA002191, 1:1,000), human DR5 (Flarebio CSB-PA08165A0Rb, 1:1,000), human DcR1 (Abcam ab133658, 1:2,000), human DcR2 (Cusabio E0425R1, 1:1,000), human caspase-3 (Beyotime AC033, 1:1,000), human caspase-8 (Beyotime AC056, 1:1,000), human caspase-9 (Beyotime AC062, 1:1,000), human cleaved-PARP (Beyotime AP102, 1:1,000), a mouse monoclonal antibody against GAPDH (Tianjin Sungene Biotech DKM9002T, 1:5,000), and IRF3 (ABclonal Q14653, 1:1,000), IRF7 (ABclonal Q92985, 1:1,000). The blots were incubated with HRP-conjugated secondary antibodies and developed by an ECL detection kit (Wuhan Goodbio Technology Co. LTD). The results were visualized using an Image Station 4000R (Kodak) and the signals were quantified using carestream software. The calculation method of protein grayscale ratio is based on the reference (37 (link)).