Cleaved caspase 3 asp175 5a1e

The endpoint xenograft tumors were isolated, fixed in formalin, and paraffin embedded. Tissue sections were processed for immunostaining and incubated overnight with p-AMPKα (T172) (40H9) (#2535, 1:200 dilution), Ki-67 (D2H10) (#9027, 1:200 dilution), or cleaved caspase-3 (Asp175) (5A1E) (#9664, 1:400 dilution) antibodies (Cell Signaling Technology). Next, the sections were counterstained with hematoxylin and subjected to IHC analysis as described previously (8 (link)).

Valproic acid and LY294002 were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in ddH₂O or dimethyl sulfoxide (DMSO) to make a stock solution at 500 mmol/L or 20 mmol/L, respectively. All the stock solutions were stored at − 20 °C. Recombinant human NRG-1 protein ab50227 was product from abcam (Cambridge, MA, USA).
MISSION® Non-target shRNA, which does not target human and mouse genes, control vector (pLKO.1-ConshRNA), and pLKO.1 containing human ErbB3 shRNA (pLKO.1-ErbB3shRNA) were purchased from Sigma. The packaging plasmids psPAX2 and pMD2.G for lentiviral expression vector were from Addgene Inc. (Cambridge, MA, USA).
Antibodies were obtained as follows: EGFR, ErbB2, ErbB3, PARP, Cleaved Caspase-3 (Asp175) (5A1E), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, STAT3, P-STAT3 (Tyr705), p21, Cyclin D1, RAS, Ki67 (Cell Signaling Technology, Inc., Beverly, MA, USA); β-actin (AC-75) (Sigma). All other reagents were purchased from Sigma unless otherwise specified.

Western blot was performed as previously described (37 (link)). The following primary antibodies, which were purchased from Cell Signaling Technology, were used: Cleaved Caspase-3 (Asp175) (5A1E) (#9664), Cleaved Caspase-8 (Asp391) (18C8) (#9496), p21 (#2947), p27 (#3686), AKT (pan) (40D4) (#2920), Phospho-AKT (Ser473) (D9E) XP (#4060), and Bim (#2933). EGFR (#18986-1-AP), LXR-α (#14351-1-AP), LXR-β (#60345-1-Ig), CHOP (#15204-1-AP), FOXO3A (10849-1-AP), and GAPDH (#60004-1-Ig) were purchased from Proteintech. FLAG was obtained from Sigma-Aldrich (# F1804). All secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States).

Antigen retrieval was performed using citrate buffer (10 mM, pH 6) or high-pH antigen retrieval solution (Vector). Sections were blocked in 10% goat serum and incubated with antibodies overnight at 4°C followed by fluorophore-conjugated antibody for immunofluorescence or HRP-conjugated secondary antibodies (Vector) for immunohistochemistry. Antibodies used were phospho DNA-PKcs-S2056 (Abcam) and phospho-histone H2AX Ser139 (γH2AX, 20E3, Cell Signaling Technology) on human and mouse tissue; RAD51 (14B4, Genetex), T1α (clone 8.1.1, DHSB), and cleaved caspase 3 Asp175 (5A1E, Cell Signaling Technology) on mouse tissue; and T1α (NC-08, Biolegend) on human tissue. Nuclei were counterstained with DAPI where appropriate. Images were acquired using a DeltaVision fluorescence microscope (Applied Precision).

Protein lysates were prepared in RIPA buffer (50 mM Tris-HCl pH7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF). Protein (10–25 µg) was electrophoresed on 10% Bis Tris or 4–12% Bis-Tris gradient NuPAGE Novex protein gels (Life Technologies) and transferred to PVDF membrane (Merck Millipore, Billerica, MA, USA). Membranes were blocked in 5% skim milk in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween-20) for 1 h before incubation with primary antibodies diluted in 5% skim milk in TBST overnight a 4 °C, or for 1 h at room temperature in the case of pan-ACTIN. All antibodies, p53 (1C12; Cell Signaling 2524), Phospho-CREB ((Ser133) Cell Signaling Technologies, #9198), Anti-CREB1 ChIP grade (ab31387), and CBP (C-1, Santa Cruz, sc-7300) were used at 1:2000, except pan-actin (Ab-5, Thermo Scientific, Waltham, MA, USA) that was used at 1:3000. Cleaved caspase 3 Asp175 [5A1E] was from Cell Signaling Technologies 9664 S. Following four 10 min washes with TBST, membranes were exposed to ECL Prime (GE Healthcare Life Sciences, Piscataway, NJ, USA) and exposed to X-ray film to detect the expression levels of proteins.