Mouse anti p21

Cis-diammineplatinum dichloride (CDDP), doxorubicin hydrochloride (DXR) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO).
Monoclonal rat anti-Gal-3 antibody was isolated from the supernatant of hybridoma (catalog number: TIB-166, American Type Culture Collection, Manassas, VA); customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Carlsbad, CA); mouse anti-V5 was purchased from Invitrogen; polyclonal rabbit anti-Bax, mouse anti-Bcl-xl, mouse anti-p-ERK, mouse ERK1/2 and rabbit anti-p-Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); mouse anti-β-actin was purchased from Sigma-Aldrich; monoclonal mouse anti-Bax was purchased from BD Biosciences (San Diego, CA); mouse anti-p21, rabbit anti-Bcl2, rabbit anti-PARP (9542L), rabbit anti-cleaved caspase-3, rabbit anti-Akt and mouse anti-caspase-8 were purchased from Cell Signaling Technology (Beverly, MA); purified rabbit IgG was purchased from ZYMED (San Francisco, CA);

Proteins were extracted by incubation with RIPA buffer and quantified by Bradford reagent. Twenty-five micrograms of protein were separated on Nupage 4–12% polyacrylamide gels (Thermo Fisher Scientific) and transferred to polyvinylidene difluoride membranes (PVDF, GE Healthcare Life Science, Piscataway, NJ, USA) to be probed with the following antibodies: mouse anti-MDM2 (1 : 500, Abcam, Cambridge, UK); mouse anti-p21 (1 : 1000, Cell Signaling, Danvers, MA, USA), and rabbit anti-β-actin (1 : 5000, Sigma-Aldrich). For detection, goat anti-rabbit or goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (1 : 2000, GE Healthcare Life Science) were used. Signal detection was performed via chemiluminescence reaction (ECL, GE Healthcare Life Science). WB quantification was performed using ImageJ software analysis (National Institutes of Health, Bethesda, MD, USA).

Protein extraction, quantification, and immunodetection were performed as in [16 , 21 (link)]. Membranes were incubated O/N with the corresponding primary antibody dilutions: mouse anti-β-ACTIN (1:5000, A5441, Sigma-Aldrich, San Luis, Missouri, USA), goat horseradish peroxidase (HRP)-conjugated anti-GAPDH (1:3500, sc-365,062, Santa Cruz, Dallas, Texas), rabbit anti-ATG4A (1:1000, #7613, Cell Signaling, Danvers, Massachusetts, USA), rabbit anti-LC3B (1:3000, 51,520, Abcam, Cambridge, UK), rabbit anti-MSI2 antibody (1:1000, EP1305Y, Abcam, Cambridge, UK), mouse anti-P62 (1:1000, 65,416, Abcam, Cambridge, UK), mouse anti-P21 (1:2000, #2946, Cell Signaling, Danvers, Massachusetts, USA), mouse anti-SCD1 (1:1000, 19,862, Abcam, Cambridge, UK). After three washes with 1x PBS-T, membranes were incubated for 1 h at RT with the corresponding secondary antibody dilutions: goat HRP-conjugated anti-rabbit-IgG (1:3500, A0545, Sigma-Aldrich, San Luis, Missouri, USA) or goat HRP-conjugated anti-mouse-IgG (1:5000, B7264, Sigma-Aldrich, San Luis, Missouri, USA). Images were acquired with an ImageQuant LAS 4000 or AMERSHAM ImageQuant 800 (GE Healthcare) and were quantified using ImageJ software (NIH).

All sample protein extracts were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking for 2 h in Tris-buffered saline with 0.1% Tween^® 20 detergent (TBST) containing 5% nonfat milk, the membranes were incubated with primary antibodies diluted in TBST containing 5% nonfat milk at 4 °C overnight. The following primary antibodies were used: mouse anti-glyceraldehyde 3-phosphate dehydrogenase (PMK Biotechnology, Shijiazhuang City, China); mouse anti-cyclinD1 (Cell Signaling Technology, Danvers, MA, USA); mouse anti-CDK4 (Cell Signaling Technology); mouse anti-P21 (Cell Signaling Technology); mouse anti-Bax (Cell Signaling Technology); and mouse anti-caspase-3 (Abcam, Shanghai, China). The membranes were washed 3 times with TBST and incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. They were then washed 3 times with TBST and the signals were visualized using ECL plus reagents (Amersham Biosciences, Buckingham, UK).