Anti acetylated histone

The changes in protein expression induced by the combination were evaluated using Western blotting. The cells were treated under the indicated conditions for 48 h, and whole-cell lysates were obtained using radioimmunoprecipitation assay buffer. Equal amounts of proteins were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After the membranes were blocked by 5% skimmed milk, they were incubated overnight with anti-acetylated histone (Abcam, Cambridge, UK), anti-cyclin D1, anti-glucose-regulated protein 78 (GRP78), anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-HDAC6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-cleaved poly(ADP-ribose) polymerase (PARP), anti-endoplasmic oxidoreductin-1-like protein alpha (Ero1-Lα) (Cell Signaling Technology, Danvers, MA, USA), or anti-actin (Millipore, Billerica, MA, USA) primary antibodies. They were then incubated with horseradish-tagged secondary antibodies (Bio-Rad, Hercules, CA, USA). The bands were visualized by chemiluminescence with the ECL Plus system (GE Healthcare, Wauwatosa, WI, USA) according to the manufacturer’s instruction.

After treatment under the indicated conditions, cells were washed with PBS, suspended in radioimmunoprecipitation (RIPA) buffer, incubated on ice for 15 min, and centrifuged at 20380 g for 12 min. Whole cell lysate was subjected to Western blot analysis as described previously.7 To analyze the expression of ubiquitinated proteins in the detergent‐insoluble fractions (pellets obtained after the protein extraction using RIPA buffer), the pellets were washed with PBS, lysed using Extraction buffer 4 in the WSE‐7421 EzSubcell Extract kit (ATTO, Tokyo, Japan), and then subjected to Western blot analysis. The following antibodies were used: anti‐cyclin D1, anti‐cyclin‐dependent kinase (CDK) 4, anti‐glucose‐regulated protein (GRP) 78, anti‐ubiquitin, anti‐histone deacetylase (HDAC) 1, anti‐HDAC3, and anti‐HDAC6 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti‐cleaved poly(ADP‐ribose) polymerase (PARP), anti‐HSP70, anti‐endoplasmic reticulum resident protein (ERP) 44, and anti‐endoplasmic oxidoreductin‐1‐like protein (Ero1‐L) from Cell Signaling Technology (Danvers, MA, USA); anti‐active caspase 3, anti‐NOXA, and anti‐acetylated histone from Abcam (Cambridge, UK); and anti‐actin from Millipore (Billerica, MA, USA).

Cells were treated under the indicated conditions for 48 hours and whole cell lysates were obtained using RIPA buffer. Tumor specimens harvested from mice were homogenized using RIPA buffer, and whole cell lysates were obtained. Equal amounts of protein were separated by 12.5% SDS‐PAGE and transferred to nitrocellulose membranes. After the membranes were blocked by 5% skimmed milk, they were incubated overnight with the primary Abs: anti‐AMPK from Proteintech; anti‐phospho‐AMPK, anti‐phospho‐S6, anti‐S6, anti‐phospho‐eukaryotic translation initiation factor 4E‐binding protein 1 (p‐4EBP1), anti‐4EBP1, and anti‐endoplasmic reticulum resident protein (ERp) 44 from Cell Signaling Technology; anti‐glucose‐regulated protein (GRP) 78, anti‐cyclin D1, anti‐cyclin‐dependent kinase (CDK) 4, anti‐HDAC1, anti‐HDAC3, and anti‐HDAC6 from Santa Cruz Biotechnology; anti‐acetylated histone from Abcam; and anti‐actin from Millipore. Then the protein was detected by reaction with HRP‐tagged goat anti‐mouse or goat anti‐rabbit Ab (Bio‐Rad) and staining with chemiluminescence solution by using the ECL Plus system (GE Healthcare).