Xhoi

Full-length wild-type Duoxa1 was amplified using PCR from mouse cDNA and ligated into the pMX-IRES-EGFP vector as pMX-Duoxa1 using the BamHI and XhoI (Enzynomics, Daejeon, Korea) sites. The following primers were used: Duoxa1-For, 5′-GCTAGGATCCATGGCTGCTCTTGGACACAC-3′ and Duoxa1-Rev, 5′-CGACTCGAGCAGGGAACAGTCGGACTCTTTG-3′. TRIzol reagent was obtained from Life Technologies (Carlsbad, CA, USA). A monoclonal β-actin (A5441) antibody and DAPI (D9542) were obtained from Sigma (St. Louis, MO, USA). Antibodies for anti-phospho-ERK-1/2, anti-total ERK-1/2, anti-phospho-p38, anti-total p38, anti-phospho-JNK, anti-total JNK, anti-phospho-IκB, anti-phospho-Akt, anti-Akt, anti-phospho-Src, anti-Src, and anti-Btk were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-Phospho-Btk (GTX61791) antibody was obtained from GeneTex (Irvine, CA, USA). Anti-c-Fos, anti-NFATc1, anti-IκB, and anti-PLCγ2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Duoxa1 antibody was obtained from Bioss Inc (BS-11433^®, Wobun, MA, USA). Donkey anti-rabbit and anti-mouse immunoglobulin secondary antibodies were purchased from Enzo Life Sciences (Farmingdale, NY, USA).

For the generation of enhanced green fluorescent protein-LC3 (EGFP-LC3) transfection vector, the full length of human microtubule-associated protein 1 light chain 3 (LC3; GenBank accession no. NM_022818.5) was amplified by RT-PCR using forward (5′-GTTCTCGAGCTATGCCGTCGGAGAAGA-3′) and reverse (5′-AAGGATCCTTACACTGACAATTTCATCC-3′) primers, and the SK-N-BE(2) cDNA was used as template. The PCR fragment of LC3 protein was digested using with XhoI and BamHI (Enzynomics, Daejeon, Korea) and was cloned into pEGFP-C1 plasmid (Clontech, Palo Alto, CA, USA). All constructs were verified by sequencing. The mRFP-GFP-LC3 expression plasmid was a generous gift from Jaerak Chang (Ajou University, Suwon, Korea).