WB
Western blot analysis of ATF6 on different lysates with Rabbit anti-ATF6 antibody at 1/1000 dilution.
Lane 1: HeLa cell lysate,
Lane 2: A431 cell lysate,
Lane 3: HUVEC cell lysate,
Lane 4: 293T cell lysate,
Lane 5: HepG2 cell lysate,
Lane 6: RAW264.7 cell lysate,
Lane 7: PC-12 cell lysate,
Lane 8: Mouse pancreas tissue lysate,
Lane 9: Mouse kidney tissue lysate,
Lane 10: Rat pancreas tissue lysate,
Lane 11: Rat kidney tissue lysate,
Lysates/proteins at 20 µg/Lane.
Exposure time: 2 minutes; 4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/1000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50000 dilution was used for 1 hour at room temperature.
IHC
Immunohistochemical analysis of paraffin-embedded rat pancreas tissue with Rabbit anti-ATF6 antibody at 1/1000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody at 1/1000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ChIP
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with 1μM Thapsigargin for 1 hour with ATF6 or Normal Rabbit IgG according to the ChIP protocol. The enriched DNA was quantified by real-time PCR using indicated primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.