Calpastatin

Antibodies directed against calpain-1 (ab39170), calpain-2 (ab39165), GAPDH (ab8245) and tubulin beta III (ab7751) were obtained from Abcam; Calpastatin from Santa Cruz (sc376547), cleaved caspase-3 (Asp175) (5A1E) (#9664) and phospho-histone H2A.X (Ser139) (#2577) from Cell Signaling Technology, TP53 (21,891–1-AP) from Proteintech, and 53BP1 clone BP13 (MAB3802) from Sigma-Aldrich. Secondary horseradish peroxidase (HRP) conjugated anti-mouse (#1,721,011) and anti-rabbit (#1,721,019) antibodies were obtained from BIO-RAD Laboratories. The AF555 anti-rabbit (ab150078) and AF647 anti-mouse (ab150115) antibodies were obtained from Abcam.

Cells were lysed in SDS sample buffer, separated by SDS-PAGE, transferred to nitrocellulose and serially probed with antibodies to Gas2 (Proteintech, Rosemont, IL), Survivin (Proteintech), Xiap1 (Proteintech), Calpastatin (Santa Cruz Biotechnology, Santa Cruz, CA), Icsbp (Santa Cruz Biotech), βcatenin (BD Biosciences, San Jose, CA) or Gapdh (as a loading control). Experiments were repeated at least twice with different lysates and representative blots are shown.

Differentiation of THP-1 and MM-6 cells was performed using Phorbol 12-myristate 13-acetate (PMA; Merck Millipore) or 1-α, 25-dihydroxyvitamin D₃ (VitD3; Sigma Aldrich). For the inhibition of kinases and proteases, the following inhibitors were applied: S6K1 inhibitor DG2 (S6K-I), PKR inhibitor (PKR-I; 6,8-Dihydro-8-1H-imidazol-5-ylmethylene)-7H-pyrrolo[2,3-g]benzothiazol-7-one), ALLN (N-[N-(N-Acetyl-L-leucyl)-L-leucyl]-L-norleucine), MG-132, Calpain-Inhibitor V (Merck Millipore), rapamycin (mTOR-I; Sigma Aldrich), UO126 (MEK-I; Cell Signaling, Danvers, USA), BI-D1870 (RSK-I; Axon Medchem BV, Groningen, Netherland), and Cathepsin-Inhibitor I (Applichem). For Western Blotting, specific antibodies directed against RSK, phosphorylated (p-)RSK (T573), ERK1/2, p-ERK1/2 (T202/Y204), rpS6, p-rpS6 (S335/236), eIF4B, p-eIF4B (S422), eIF4A, eIF2α, p-eIF2α (S51), PKR (Cell Signaling), p-PKR (T446; Abcam, Cambridge, UK), V5 Tag (Invitrogen), C/EBPβ, Calpastatin, Histon H4 (Santa Cruz), Actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or TATA box-binding protein (TBP; Sigma Aldrich) were used in combination with secondary horseradish peroxidase (HRP-)coupled antibodies (Vector Laboratories, Peterborough, UK). All media and reagents were of the best available grade and routinely tested for endotoxins with the Limulus Amoebocyte Lysate assay (Lonza).