Ecl hrp substrate

Frozen mouse tissues were homogenized in CelLytic M cell lysis reagent (MilliporeSigma) and cOmplete protease inhibitors (Roche) was added to prevent protein degradation. Total protein concentration of the supernatants from centrifuged tissue lysates was determined by BCA protein assay (Pierce). Eight micrograms of total protein lysate were resolved on 4–15% Mini-PROTEAN TGX Stain-free gels (Bio-Rad) and transferred onto Immuno-Blot PVDF membranes (Bio-Rad). Membrane blots were blocked with EveryBlot blocking buffer (Bio-Rad) and probed with an anti-LC3B primary antibody (cat# L7543, Sigma) followed by an HRP-conjugated secondary antibody before applying ECL HRP substrate (Bio-Rad) for chemiluminescence. Stain-free gels and blots were imaged using the stain-free and chemiluminescence settings on the ChemiDoc™ MP imaging system (Bio-Rad). LC3B-I and LC3B-II protein levels were measured by densitometric analysis of western blots using Fuji software (ImageJ version 2.0)^{38 (link)}. Signals were normalized to the amount of total protein as determined by densitometric analysis of stain-free gels. LC3B-II/LC3B-I ratio was normalized to WT for each organ.

The cells were lysed with HEPES lysis buffer supplemented with a protease inhibitor cocktail (#HY-K0010, MedChemExpress, Monmouth Junction, NJ, USA) and Na₃VO₄, a phosphatase inhibitor. The protein concentration was determined using the Bradford protein assay (Bio-Rad, Hercules, CA, USA). The proteins were separated by 8–12% SDS-PAGE and transferred onto the PVDF membrane. The membranes were blocked with 5% BSA in TBS-N for 1 h with these following primary antibodies: pY-STAT3 (Y705) (#9131S), caspase-3 (#9662S), vimentin (D21H3) (#5741T), E-cadherin (24E10) (#3195T), N-cadherin (D4R1H) (#13116T), claudin-1 (D5H1D) (#13255T), β-catenin (D10A8) (#8480T), β-actin (#4967) (Cell Signaling Technology, Inc. (Danvers, MA, USA)), and STAT3 (#610190) (BD Biosciences, San Jose, CA, USA) at 1:1000 dilution; then the membranes were incubated overnight at 4 °C. Membranes were washed three times with TBS-N and then incubated with secondary antibody (mouse: #ab6789, rabbit: #ab6721) (both from Abcam, Cambridge, UK) for 30 min. After washing, the immunoblots were visualized using a chemiluminescence (ECL) HRP substrate (Bio-Rad, California, USA). The data were analyzed via densitometry using ImageJ software and normalized to the expression of the internal control (β-actin).

U87-MG and LN229 cells were treated with C-10, MJ, and 2-DG molecules at 5 mM for 24 h to determine the expression levels for apoptotic and necrotic markers. The cells were incubated at +4°C on the shaker for 30 min using RIPA lysis buffer with a protease inhibitor. The pellets of all samples were then collected. The total protein samples were then treated with 2X Laemli buffer (BioRad). All protein samples were loaded in a 4–15% gel at an equal concentration (25 µg) and run in an electrophoresis tank. The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes using the Trans-Blot TurboTransfer System (BioRad). Following transfer, the membranes were shaken in 5% non-fat milk dissolved in 1X TBST (137 mM NaCl, 20 mM Tris, 0.1% Tween-20) RT for 1 h. The membranes were incubated with primary antibodies overnight at +4°C (anti-LC3 (Sigma #L8918) (1: 1,000). The next day, the membranes were washed three times with 1X TBST for 5 min and then with an HRP-conjugated secondary antibody (anti-rabbit CST # 7074S) (1: 2000) 5% non-fat milk dissolved in 1X TBST (137 mM NaCl, 20 mM Tris, 0.1% Tween-20) incubated at room temperature for 1 h. The protein bands were detected by ChemiDoc MP System (Bio-Rad) using the ECL HRP substrate (Biorad). The protein levels were analyzed using ImageJ, and the β-actin bands were used for normalization.