Ripa assay buffer

Cells subjected to IL-8 siRNA were harvested and lysed in radioimmunoprecipitation (RIPA) assay buffer (Santa Cruz Biotechnology, USA) including 1% phosphatase inhibitor, protease inhibitor and sodium orthovanadate. Protein lysates were run on SDS-polyacrylamide gels and blotted onto nitrocellulose membrane (Pall Corporation, USA). Following transfer, membranes were blocked in 5% bovine serum albumin (BSA) in TBS-Tween, probed with antibodies for HSP60, Bcl-2 or p-Bad S136 at a dilution of 1:1000 in 5% BSA/TBS-T overnight at 4°C, washed three times in TBS-T, incubated in corresponding secondary antibodies for 1 hour at room temperature, washed three times, and bound antibodies visualized by Immobilon enhanced chemiluminesence (Millipore, UK).

The total protein of cell or tissue lysate was isolated as previously described [45 (link)]. Cells were harvested in radioimmunoprecipitation (RIPA) assay buffer(Santa Cruz Biotechnology,#sc-24948). Protein content was quantified using the Bradford assay with BSA as standard (Bio-Rad), and 30μg of total protein lysate were separated on SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking (5% non-fat milk in PBS, 1 h at RT), membranes were incubated at 4°C overnight with the following primary antibodies: Hsp60 antibody (Affinity, #AF0184,1:1000), β-actin monoclonal antibody (Kangchen, #KC-5A08,1:5000), AFP antibody(Abcam, #3980, 1:1000), E-cadherin antibody (proteintech,# 20874-1- -AP,1:1000), Vimentin Antibody (proteintech, #10366-1-AP,1:1000), N-Cadherin antibody (Abcam, #ab18203) and COX4 antibody (Abgent, #ALS12730,1:1000). After three washes with PBS, membranes were incubated with peroxidase-labelled secondary antibodies (Merck Millipore, 1:5000) at 37° C for 1 h. Immunoblots were developed using the enhanced chemiluminescence reagent (Pierce, #32209) .

Cells cultured in the 3D-collagen gel were recovered following enzymatic dissociation with collagenase 1 (2 mg/mL) and whole cell lysates were prepared by incubation with radioimmunoprecipitation (RIPA) assay buffer (Santa Cruz) supplemented with a cocktail of protease inhibitors (Santa Cruz) and the total protein concentrations were determined using bicinchoninic acid (BCA) method (BioVision). Then 30 µg of proteins were subjected to SDS-polyacrylamide gel electrophoresis before being electroblotted onto a 0.2 μm nitrocellulose membrane (GE Healthcare). After blocking with 5% nonfat dry milk in TBST [25 mmol/L Tris (pH, 7.4), 137 mmol/L NaCl, 0.5% Tween20], membranes were incubated at 4 °C overnight with following primary antibodies: p75 (1:1000, Cell Signaling), NOCTH3 (1:1000, Abcam), HES1(1:1000, Cell Signaling). GAPDH (1;2000, Cell Signaling) was used as a loading control. After extensively washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz), and blot signals were developed with ECL^TM Western Blotting Detect Reagents (GE Health Care). All blots were derived from the same experiment and processed in parallel. Uncropped Western blotting images were provided in Supplementary Fig. 8 with the size markers labeled.