Cst2729

The immunoprecipitation protocol used was described previously [28 (link)]. Briefly, the immunoprecipitation buffer (150 mM NaCl, 10 mM Hepes, 2 mM EDTA, 1% Triton, 1.5 mM MgCl2, 10% glycerol, EDTA-free Protease Inhibitor Cocktail (4693159001 Roche), PhosSTOP^TM (4906845001 Roche)) was used to lysate the cells. Cells were then centrifuged and supernatants were incubated at 4 °C overnight with magnetic beads (161–4021, Bio-Rad, Madrid, Spain) previously conjugated to 5 μg of rabbit anti-MCL-1 antibody (CST94296, Cell Signaling, Leiden, The Netherlands) or 5 μg of rabbit IgG antibody (CST2729, Cell Signaling). After magnetization, supernatant was discarded and the binding fraction was resuspended in 40 μL 4X SDS-PAGE sample buffer and heated at 70 °C for 10 min. Finally, the sample was magnetized to collect the supernatant, which was stored at −80 °C for further analysis.

Cells were lysed in immunoprecipitation buffer (150 mM NaCl, 10 mM Hepes, 2 mM EDTA, 1% Triton, 1.5 mM MgCl2, 10% glycerol and EDTA-free Protease Inhibitor Cocktail (4693159001, Roche, Mannkin, Germany)) and centrifuged at 14,000× g, 15 min at 4 °C. The resulting supernatants were incubated with magnetic beads (161-4021, Bio-Rad, Madrid, Spain) conjugated to 5 µg of rabbit anti-BIM antibody (CST2933, Cell Signaling, Leiden, The Netherlands) or 5 μg of rabbit IgG antibody (CST2729, Cell Signaling, Leiden, The Netherlands) at 4 °C overnight. A fraction of the supernatant (30 μL) was removed and mixed with half volume of 4× SDS-PAGE sample buffer, heated at 96 °C for 5 min, and stored at −80 °C as cell lysate fractions. After magnetization, a part of the supernatant was mixed with half volume of 4× SDS-PAGE sample buffer, heated at 96 °C for 5 min, and stored at −80 °C as unbound fractions. The rest of the supernatant was discarded. The resulting pellet was washed and mixed with 40 µL 4× SDS-PAGE sample buffer and heated 10 min at 70 °C to allow dissociation between the purified target proteins and the bead–antibody complex. Finally, sample was magnetized, and the supernatant was collected and stored at −80 °C as immunoprecipitation (IP) fractions for further Western blot analysis.

For chromatin immunoprecipitation (ChIP) assay, we used mouse CAF cells (97f) that were isolated from Ptf1a-^Cre/+; LSL-Kras^G12D/+;Tgfbr2^flox/flox mice, a genetically engineered mouse model of PDAC that well recapitulates desmoplastic stroma in human PDAC [49 (link)]. For each assay, sheared chromatin equivalent to 7.5 × 10⁶ cells were used. For each assay, 40 μL of magnetic beads (Dynabeads M-280 Seep anti-Rabbit IgG, Life Technologies) were blocked with 0.5 % BSA in PBS and further bound with 4 μg of indicated antibodies overnight at 4°C. Antibodies used for ChIP were: BRD4 (Bethly A301-985A), H3K27ac (Abcam, ab4729), RNA polymerase II (Santa Cruz, sc-899X), SMAD3 (Abcam, ab28379), and normal Rabbit IgG (Cell Signaling Technology, CST2729). After purification of precipitated DNA, qPCR was performed. Detailed procedures for ChIP are described in Supplementary Methods. The ChIP primers are listed in Supplementary Table S4.