High capacity cdna rt

Vitamin D analog TX 527 [19-nor-14,20-bisepi-23-yne-1,25(OH)₂D₃] was originally synthesized by M. Vandewalle and P. De Clercq (University of Ghent, Ghent, Belgium) and provided by Théramex (Monaco). Immobilon P (polyvinylidenedifluoride; PVDF) membranes, 1α,25(OH)₂D₃, the antibiotic G418 and LY294002 were from Sigma-Aldrich (St. Louis, MO, USA). Puromycin supplied by Invivogen (San Diego, CA, USA). The antibodies used were rabbit monoclonal anti-p-Akt, anti-Akt, anti-MEKα (Cell Signaling Technology, Danvers, MA, USA) and anti-Tubulin (Thermo Fisher Scientific Inc., Waltham, MA, USA); mouse monoclonal anti-LC3, anti-BECN1, anti-p-mTOR, and anti-mTOR, goat polyclonal Lamin B and secondary antibodies anti-rabbit, anti-mouse and anti-goat (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Roche Applied Science supplied high Pure RNA Isolation Kit (Indianapolis, IN, USA). High Capacity cDNA RT and SYBR Green PCR Master Mix reagent (Applied Biosystems) were acquired from Thermofisher (Thermofisher, Buenos Aires, AR). GAPDH primer was from Invitrogen^TM (Invitrogen/Thermo Fisher Scientific Inc., Waltham, MA, USA) and BECN1 primer was obtained by Eurogentec (Serain, Belgium). The inhibitor of flux autophagy, Chloroquine, was kindly provided by Dr. Daniel Grasso (IBIMOL Universidad de Buenos Aires – CONICET).

Total RNA for real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis was isolated using the High Pure RNA Isolation Kit (Roche) [34] (link). Total RNA (1 μg) was reverse transcribed using the kit High Capacity cDNA RT (Applied Biosystems, Thermofisher, Buenos Aires, AR) and qRT-PCR reactions were achieved on the resulting cDNA (5–10 ng) in an ABI 7500 Real Time PCR system (Applied Biosystems, CA, USA) using specific primers to detect A20 and BECN1 levels and GAPDH to normalize gene expression. Primers used for amplification were: murine Gapdh, forward 5′-AAGGTGAAGGTCGGAGTC-3′, reverse 5′-GAAGATGGTGATGGGATTTC-3′; murine Becn1, forward 5′-GGACAAGCTCAAGAAAACCAATG-3′, reverse 5′-TGTCCGCTGTGCCAGATG-3’; murine A20 forward 5′-CATGAAGCAAGAAGAACGGAAGA-3′, reverse 5′-GAGGCCCGGGCACATT-3’. Reactions were carried out using the SYBR Green PCR Master Mix reagent (Applied Biosystems). Gene expression was then analyzed by 2-delta delta Ct method [43] (link).