Anti mdm2

The protein used for western blot analysis was extracted using RIPA lysis buffer (Beyotime Biotechnology) containing protease inhibitors (Roche, Switzerland). Equivalent proteins (30 μg) were loaded on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% bovine serum albumin (BSA; Roche) for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4°C. The primary antibodies were prepared in 5% BSA at a dilution of 1:1000. Primary antibodies used in the study were as follows: anti-Bcl-2 (#4223), anti-Bax (#5023), anti-caspase-3 (#9662), anti-caspase-9 (#9502), anti-PARP (#9532), anti-p53 (#2524), anti-p-p53 (#9284), anti-p21 (#2947), anti-Mdm2 (#86934), anti-γ-H2AX (#9718), and anti-β-actin (#4970; Cell Signaling Technology). Then, blots were rinsed with Tris-buffered saline plus Tween-20 (TBST) and incubated with secondary antibody marked by horseradish peroxidase for 1 h at room temperature. After being rinsed, the membranes were subjected to the ChemiDocTM XRS system (Bio-Rad), and Immobilon Western Chemiluminescent HRP Substrate (Millipore) was added. The signal intensity of each band was captured and quantified using Image Lab^TM software (Bio-Rad).

Oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, antimycin, and CHX were purchased from Sigma-Aldrich (St. Louis, MO). Decitabine, monensin, MG132, KX2-391, and IACS-010759 were from Selleck (Houston, TX). Recombinant human CYTL1 was purchased from OriGene Technologies (Rockville, MD). Anti-CYTL1 antibody was purchased from Abcam (Cambridge, UK). Anti-NDUFV1, anti-BRCA1, anti-Flag, anti-GAPDH, and anti-Src antibodies were from Proteintech Group (Chicago, IL). Anti-HA, anti-CD31, anti-MDM2, anti-LDHA, anti-p-LDHA (Y10), and anti-COX4 antibodies were from Cell Signaling Technology (Beverly, MA). Anti-Tubulin, anti-β-Actin, anti-myc, and p-Src (Santa Cruz) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Lipofectamine 3000 and Lipofectamine RNAi MAX were purchased from Thermo Fisher Scientific (Waltham, MA). MitoCheck Complex I activity assay kit (No.700930) was purchased from Cayman Chemical (Ann Arbor, Michigan). Human CYTL1 ELISA Kit was purchased from CUSABIO Life Sciences (College Park, MD).

Cells were harvested and rinsed with PBS, and lyzed in denaturing lysis buffer (Applygen Technologies Inc. China) for 30 min on ice, centrifuged 12,000 g for 20 min at 4 °C. Protein concentrations were determined by BCA assay. Equal quantities (30 μg of protein) of cell extract were resolved by 10% SDS-PAGE, the resolved protein were electrophoretically transferred to PVDF membrane, and blocked with 5% fat-free dry milk in TBST for 1 h at room temperature. The membrane was immunoblotted with anti-γ-H2AX, anti-p21, anti-p-ATM, anti-ATM, anti-p-Mdm2, anti-Mdm2, anti-p-p53, anti-AKT, anti-β-actin (Cell Signaling Technology, USA), anti-p53, anti-Bax and anti-Bcl-2 (Santa Cruz, USA) antibodies in 5% milk TBST, at 4 °C overnight. The membranes were washed 3 times, incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, and washed extensively before detection. The membranes were subsequently developed using ECL (FujiFilm, Japan) reagent (Applygen Technologies Inc. China) and exposed to film according to the manufacturer’s protocol.

We performed western blot (WB) analysis as previously described³⁶ using an ECL kit (4AW011; purchased from 4A Biotech Co., Ltd) and the following antibodies: anti‐GAPDH (1:5000, 60004‐1‐Ig; Proteintech, USA), anti‐αSMA (1:1000, ab124964; Abcam, UK), anti‐fibronectin (1:1000, ab45688; Abcam, UK), anti‐MDM2 (1:1000, #86934; Cell Signaling Technology, USA), anti‐t‐p53 (1:1000, 10442‐1‐AP; Proteintech, USA), anti‐Col1 (1:1000, #84336; Cell Signaling Technology, USA), anti‐vimentin (1:1000, #5741; Cell Signaling Technology, USA), anti‐CD68 (1:5000, 25747‐1‐AP; Proteintech, USA), anti‐p‐p38 (1:1000, #4511; Cell Signaling Technology, USA), anti‐t‐p38 (1:1000, #8690; Cell Signaling Technology, USA), anti‐p21 (1:1000, ab109520; Abcam, UK), and nti‐FSP1 (1:1000, ab124805; Abcam, UK).

For Western blot analysis, cells were lysed with ice-cold Cell Lysis Buffer (Cell Signaling, Cat #9803S) supplemented with Halt™ Protease and Phosphatase inhibitor Cocktail (Thermo Scientific, Cat #1861281) and incubated at 4°C for 30 min to extract proteins. Equivalent amounts of proteins were first mixed with sample buffer (Bio-Rad, Cat #1610747), then loaded on a Tris-HCl 4%–20% precast polyacrylamide gel (Bio-Rad, Cat #4561094) and transferred onto nitrocellulose membranes (Bio-Rad, Cat #1704159). Membranes were probed overnight at 4°C with anti-MDM2 (Cell Signaling Technology Cat# 86934, RRID:AB_2784534) and anti-GAPDH antibodies (Santa Cruz Biotechnology Cat# sc-48167, RRID:AB_1563046), followed by incubation with fluorescence-conjugated secondary antibodies IRDye^® 800CW (Li-Cor, Cat #926-32213, RRID: AB_621848) and IRDye^® 680RD (Li-Cor, Cat #926-68074, RRID: AB_10956736). The proteins of interest were finally detected using Odyssey CLx Imaging System (Li-Cor). The band density of proteins was quantified using densitometric Image Studio Software Ver. 5.2 (Li-Cor Biosciences).