Bcl 2

HaCaT-ras II4 cells were treated with two different concentrations of F2 (25 and 50 μg/mL) for 48 h. Adherent and non-adherent cells were collected on ice, washed twice with PBS, lysed with lysis buffer and centrifuged at 12,000 g for 10 min at 4 °C. The cell lysate was heated at 100 °C for 5 min, and the protein content was determined by the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA).
Equal protein concentrations were subjected to Western blot analysis as described previously [20 (link)]. The PVDF membranes were blocked with blocking buffer (1× TBS, 0.1% Tween-20, 5% skim milk) for 2 h and then probed with primary rabbit polyclonal antibodies to Caspase-3, AKT, p-AKT, ERK and p-ERK (Abcam, Cambridge, UK) and mouse monoclonal antibodies to actin, p53, p21, Bcl-2, BAX (Santa Cruz, CA) at dilution ranging from 1/1000 – 1/5000 at 4 °C overnight. Later, the primary antibodies were washed away with TBST for 2 h and the membranes were treated with horseradish peroxidase (HRP)-coupled secondary antibodies in blocking buffer at a dilution of 1/5000 (Abcam, Cambridge, UK) for 1 h, and washed with TBST afterwards. Finally, detection of each protein was performed using the chemiluminescence ECL kit (Abcam plc, Cambridge, UK). Blot images were obtained with the image lab Software (BioRad, Chemidoc imaging instrument).

Cells were harvested and lysed using a RIPA lysis buffer (Santa Cruz Biotechnology) with a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). An equal amount of proteins was separated by SDS-PAGE and analyzed by immunoblotting with primary antibodies for cleaved caspase3 (Cell Signaling Technology, Danvers, MA, USA), Bax, Bcl2, and actin (Santa Cruz Biotechnology).

The cultured HT-29 cells were treated with MFGM from five species of milk at 100 μg/mL for 72 h at 37 °C, 5%CO₂. The HT-29 cells were lysed in RIPA lysis buffer (containing PMSF) for 2 h, then they were sonicated and centrifuged for 10 min at 4 °C at 12000 × g. Protein concentration in the cell lysates was determined by Lowry’s method^{68 (link)}. Proteins were separated by 10% SDS–PAGE, and transferred onto a PVDF membrane. Membranes were washed with TBST (10 mM Tris, 100 mM NaCl, 0.1% Tween 20) followed by blocking with 5% skim milk powder in TBST for 1 h at room temperature. The membranes were incubated overnight (4 °C) with specific primary antibodies (Bax, Bcl-2, Caspase-3, and β-Actin) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were washed twice with TBST, incubated with appropriate secondary antibody conjugated with HRP for 2 h at 37 °C, and the bands were visualized with an ECL chemiluminescence kit (Millipore Corporation, Billerica, MA, USA). Image J software was used to analyze gray-scale value of bands.

RIPA lysis buffer was used to lysate cells.³⁴ Western blot analysis was performed using equal quantity of protein. Protein concentration was determined by Bradford method (Sigma‐Aldrich) and equal amounts were subjected to Western blot analysis. All membranes were incubated for 12 hours at 4°C with antibodies against sirt1, ERα, IGF1R, CCND1, vimentin, N‐cadherin, bax, bcl‐2, parp1 and cytochrome c (all from Santa Cruz Biotechnology, Santa Cruz CA, USA). Membranes were incubated with HRP‐conjugated secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA), and immunoreactive bands were visualized with the ECL Western blotting detection system (Santa Cruz Biotechnology, Santa Cruz CA, USA). GAPDH (Santa Cruz Biotechnology, Santa Cruz CA, USA) or VDAC1/porin (Abcam, Cambridge, UK) antibodies were used as a loading control.

Primary antibodies against Grp78, GADD34, CHOP, p-Ire-1 α, p-PERK-1 and p-c-Jun were purchased from Cell Signaling Technology (Danvers, MA); Bax, bcl-2, bcl-x, actin and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). CaspGLOW Fluorescein Active Caspase Staining Kits were purchased from Biovision (Milpitas, CA). Cell culture media were obtained from Hyclone (South Logan, UT). DiOC₆, Fluo-3 and Rhod-2 were purchased from Invitrogen (New York, USA). Western blot chemiluminescence reagents were purchased from Millipore (Boston, MA). The lentiviral siRNAs, shGADD34 (TRCN03041) and shGFP (TRCN 072178) were purchased from National RNAi Core Facility (Taipei, Taiwan) and DNA sequence is shown in Supplemental Table 3. All other chemicals were obtained from Bio-Rad (Richmond, CA), USB (Darmstadt, Germany) or Sigma Chemical (St. Louis, MO) and were the molecular biologic grade or higher. The use of the toxic chemicals followed the rules of Toxic Chemical Substances Control Act of Taiwan. All the following experiments also followed the guidelines of Good Laboratory Practice of Taiwan.