Pcdna3

We have used human CaM and associated CaM mutants that we have described before (9 (link)), CaM(1–44) and partial CaMs: N-CaM(1–74), C-CaM(75–148), and CaMs that simulate CaM binding in the absence of calcium, by substitution of glutamate residues in the two calcium-binding sites in the N-terminal lobe (CaM₁₂) or C-terminal lobe (CaM₃₄), or mutation of all four calcium-binding sites (CaM₁₂₃₄) of CaM. CaM–GFP used for Western blotting is a construct consisting of GFP fused to the C terminus of CaM expressed in pcDNA3.1 (Cell Signaling Technology) (83 (link)). EGFP expression vector was obtained by subcloning EGFP from pEGFP-C1 (BD Biosciences) into the multiple cloning site of pcDNA3.1. The CaM₁₂₃₄ overexpression construct contained bicistronic marker mRFP (to generate a red color) (46 (link)) to distinguish from the channel expression with bicistronic EGFP (to generate a green color). The CaM₁₂₃₄ overexpression construct also containing CaM shRNA knockdown includes two short hairpin sequences described above to knock down CaM (Calm_1,2,3 genes) (46 (link)– (link)48 (link)). The shRNA knockdown sequences is reported to be 70% complete in disabling of native CaM expression (46 (link)– (link)48 (link)), but this knockdown was insufficient for our purposes, as we observed no significant physiological consequence after transfection of the shRNA CaM knockdown alone.

For immunoblot and immunohistochemical analysis, various antibodies like rabbit monoclonal anti-p-MAPK, anti-MAPK, anti-p-Akt (Ser & Thr) and anti-Akt, anti-p-GSK-3β, anti-GSK-3β, anti-p-FoxO3a, anti-FoxO3a monoclonal mouse anti-CD-31, rabbit anti-caspase 3/7 were purchased from Cell Signaling Technology, Beverly, MA, USA. Mouse anti-Ki-67, rabbit monoclonal anti-PARP, mouse monoclonal anti-Bcl-2, anti-Bax, anti-Mcl-1, anti-Bcl-xL, HRP conjugated goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Mouse monoclonal anti-β-actin and other reagents were procured from Sigma Aldrich, St. Louis, MO, USA. Antibodies dilutions have been made as per the manufacturer’s instruction. The pcDNA3-Akt-HA plasmid was obtained as gift sample from Dr. Guy Salvesen (University of California, San Diego, CA, USA). siRNA Akt and pcDNA3.1(-) were purchased from Cell Signaling Technology, Beverly, MA, USA and Invitrogen, USA respectively. Opti-MEM^®I Reduced Serum Media and fetal bovine serum (FBS) were purchased from Gibco-BRL Invitrogen Corporation, CA, USA. FuGENE® HD transfection reagent was obtained from Roche Applied Science, Mannheim, Germany. Trypsin, Bovine serum albumin (BSA) and antibiotics (10,000 U/L penicillin and 10 mg/L streptomycin) were purchased from Himedia, Mumbai, India.