Mcl 1

Total cellular protein was isolated from the cells after various treatments. For Western blots, a previously described procedure was applied [54 (link)]. The following primary antibodies were used: Acetyl Histone H3, HDAC1, HDAC8, PPARγ, cyclin D1, CDK6, p-Ser⁴⁷³ Akt, Akt, p-Ser²⁴⁴⁸ mTOR, mTOR, p-Ser¹³⁹ H2AX, H2AX, Bax, Mcl-1, PARP, procaspase-8, cleaved caspase-9, LC3B, and Atg5 were purchased from Cell Signaling Technologies (Beverly, MA, USA); β-actin, Sigma-Aldrich (St. Louis, MO, USA). The secondary antibodies were purchased from Santa Cruz Biotechnology. The enhanced chemiluminescence (ECL) system for detection of immunoblotted proteins was from GE Healthcare Bioscience (Piscataway, NJ, USA). Then, the protein was visualized by FUSION SOLO S (VILBER, Deutschland, Germany).

After the indicated treatment, cells were harvested and lysed in lysis buffer. Equal amount of proteins (10–30 μg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Millipore). The membranes were then blocked with 5% non-fatty dry milk in TBST for 2 h and probed overnight at 4°C with primary antibodies against the following proteins: 14-3-3ζ (1/1000, Abcam), E-cadherin (1/1000, Cell Signaling Technology), vimentin (1/1000, Cell Signaling Technology), Snail (1/1000, Cell Signaling Technology), Slug (1/1000, Cell Signaling Technology), Bcl-2 (1/1000, Cell Signaling Technology), Bcl-xL (1/1000, Cell Signaling Technology), MCL-1 (1/1000, Cell Signaling Technology), Bax (1/1000, Cell Signaling Technology), PARP (1/1000, Cell Signaling Technology), cleaved caspase-3 (1/1000, Cell Signaling Technology) and β-actin (1/2000, Cell Signaling Technology). The membranes were washed in TBST and then incubated in HRP-conjugated secondary antibodies (1/5000, Cell Signaling Technology) for additional 1 h. The specific protein bands on the membranes were visualized by enhanced chemiluminescence (Millipore).

For Western blotting, total protein extracts were obtained in cell lysis buffer containing 150 mM NaCl, 1 mM EDTA, 1% NP-40, 50 mM Tris (pH 8.0) as well as phosphatase and protease inhibitors. Protein concentrations were determined by BCA staining (#23225, Thermo Fisher Scientific, Hennigsdorf, Germany) as compared to BSA concentration standard. Following SDS polyacrylamide gel electrophoresis, proteins were blotted on nitrocellulose membranes, as described previously [55 (link)].
Several primary antibodies were derived from Cell Signaling Technology (Danvers, MA, USA): caspase-3 (9662, rabbit, 1:1000), caspase-8 (9746, mouse, 1:1000), caspase-9 (9502, rabbit, 1:1000), Mcl-1 (4572, rabbit, 1:1000), Bcl-w (2724, rabbit, 1:1000) and Bcl-2 (2872, rabbit, 1:1000). Other primary antibodies were derived from Santa Cruz Biotech (Dallas, TX, USA): Bcl-x_L (sc-8392, mouse, 1:1000) and β-actin (sc-47778, mouse, 1:1000). As secondary antibodies, peroxidase-labeled goat anti-rabbit and goat anti-mouse were used (Dako, Hamburg, Germany; 1:5000).

Immunoblotting was performed with the following antibodies: Bax (D2E11, #5023), Bcl-xL (#2762), Caspase-3 (#9662), Caspase-9 (#9502), p-Cdc2 (Tyr15) (#9111), Cdc2 (POH1, #9116), p-CDK2 (Thr160) (#2561), p-Chk2 (Thr68) (C13C1, #2197), Chk2 (#2662), c-Raf (#9422), CDK2 (78B2, #2546), Cyclin D1 (#2922), Cyclin E1 (D7T3U, #20808), DUSP4 (D9A5, #5149), DUSP6 (#39441), p-Erk1/2 (Thr202/Tyr204) (D13.14.4E, #4370), Erk1/2 (137F5, #4695), Mcl-1 (D5V5L, #39224), MDM2 (D1V2Z, #86934), p-MEK1/2 (Ser217/221) (41G9, #9154), MEK1/2 (47E6, #9126), p21 (12D1, #2947), p-p53 (Ser15) (16G8, #9286), p53 (7F5, #2527), PARP (46D11, #9532), PUMA (D30C10, #12450), p-Rb (Ser789) (#9307), (all from Cell Signaling Technology (Boston, MA, USA)), Actin (C-2, #sc-8432 AF790, Santa Cruz Biotechnology), and β-actin antibody (AC-15, #A5441, Sigma-Aldrich). Immunoblotting signals were visualized using an Odyssey IR imaging system (Li-Cor Biosciences, Lincoln, NE, USA). Image Studio software v4.0 was used for analysis of the bands.

For western blotting, cells were lysed buffer including 1% Triton X-100 and 1% NP-40, as well as the following protease and phosphatase inhibitors. The first step is to separate the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then, proteins can be electrophoretically transferred to PVDF membranes. The membranes were immunostained with primary antibodies. Furthermore, HRP-conjugated secondary antibodies are added. Detection was carried out using an enhanced chemiluminescence reagent (Amersham Biosciences, Buckinghamshire, UK). Antibodies against cleaved PARP, cleaved caspase-3, XIAP, Mcl-1, p-mTOR, p-AKT (Tyr308), p-4EBP1, p-GSK3β, and β-actin were purchased from Cell Signaling Technology (Danvers, MA), Abcam (Cambridge, MA), and Santa Cruz Biotechnology (Santa Cruz, CA).

For immunoblotting and co-immunoprecipitation: Actin (Santa Cruz, ♯sc-47778; 1:5000), ATF6 (Cell Signaling Technology, ♯65880; 1:1000), BCL-2 (AbCam, ♯ab182858; 1:500), BCL-XL (Cell Signaling Technology, ♯2764; 1:1000), Cleaved-Caspase-8 (Cell Signaling Technology, ♯8592; 1:1000), Caspase-3 (Cell Signaling Technology, ♯9665; 1:1000), Caspase-9 (Cell Signaling Technology, ♯9508; 1:1000), CHOP (Cell Signaling Technology, ♯2895; 1:250), GFP (AbCam, ♯ab13970; 1:2000), GRP78 (Cell Signaling Technology, ♯3177; 1:1000), FLAG-HRP (Sigma Aldrich, ♯A8592; 1:4000), HA-HRP (Roche, ♯11867423001; 1:2000), IP₃R1 (Thermo Fischer, ♯PA1-901; 1:1000), IP₃R1 & IP₃R2 [previously described⁷⁷ ; 1:1000], IP₃R3 (BD Biosciences, ♯610312; 1:1000), KDEL (AbCam, ♯ab12223; 1:2000), MCL-1 (Cell Signaling Technology, ♯5453; 1:1000), Nicastrin (BD Biosciences, ♯612290; 1:1000), PARP (Cell Signaling Technology, ♯9542; 1:1000), SERCA2 (Cell Signaling Technology, ♯4388; 1:1000), STIM1 (Cell Signaling Technology, ♯5668; 1:1000).
For immunofluorescence: DAPI (Thermo Fischer, ♯D1306; 1 µg/ml), HA (Cell Signaling Technology, ♯3724; 1:500), BAP31 (Enzo Life Sciences, ♯ALX-804-601-C100; 1:250).
For proximity ligation assay: HA (Cell Signaling Technology, ♯3724; 1:500), HA (Enzo Life Sciences, ♯ENZ-ABS118-0200; 1:200), IP₃R1 (Thermo Fischer, ♯PA1-901; 1:200), IP₃R3 (BD Biosciences, ♯610312; 1:200).