FoxP3 (Abcam, Cambridge, MA, USA), CD86 and CD80 (R&D systems) and actin (Santa Cruz Biotechnology, Dallas, TX, USA) levels in cell extracts were detected using the respective primary and secondary antibodies and detected using the chemiluminescent horseradish peroxidase system (Pierce, Rockford, IL, USA) as described previously (Garg et al., 2006).
Expression of specific proteins was assessed using the respective antibodies: Smad4 (Oncogene, Boston, MA, USA), caspase-3 (Pharmigen, San Diego, CA, USA), I κB α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TIEG1 (a generous gift from Dr R Urrutia, Mayo Clinic, Rochester, MN, USA) and α-actin was from Calbiochem #CP01; San Diego, CA, USA).
Amplicons were purified by the DNA Clean and Concentrator Kit (Zymo Research, Irvine, USA) and subsequently sequenced by Eurofins MWG Operon (Ebersberg, Germany) using the respective forward primers.
For analysis of membrane phosphatidylserine exposure, phosphatidylserine switch to the outer leaflet of the plasma membrane was analyzed by Annexin-V staining using the respective apoptosis detection kit (ebioscience, San Diego, CA, USA) according to the manufacturer's instructions.
The activities of β-glucosidase, β-xylosidase, β-mannosidase, α-L-arabinofuranosidase, and cellobiohydrolase II were measured using the respective p-nitrophenol residues (pNP) (Sigma-Aldrich, USA).
The ELISA was performed using the respective secondary antibodies.
The metal content (viz. Ca, Fe, Zn, Mg) of the sample extract was analyzed through Atomic Absorption Spectrometer (AAS, Aanalyst 200, Perkin Elmer, USA) using respective hollow cathode lamps (HCL, Perkin Elmer).
Of the resulting cDNA, 30 ng/μl was used as the template to quantify the relative content of mRNA by real-time PCR (ABI PRISM 7700 sequence detection system, Applied Biosystems, Foster City, CA USA) using respective primers and SYBR Green.
Scoring of fragment sizes and fluorescence intensity/peak heights (in tetraploids) was automatically performed with GeneMarker version 1.95 (SoftGenetics, State College PH, USA) using respective panels for each locus with subsequent manual checking of each sample.
Forward and reverse sequencing of clones was conducted on ABI 377 and 3700 DNA sequencers (Applied Biosystems, Foster City, CA, USA), using, for each respective sequencer, a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences, Piscataway, NJ, USA) and an ABI BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).
