Western blot analysis of ATF-4 on different lysates with Rabbit anti-ATF-4 antibody (HA722796) at 1/2,000 dilution. Lane 1: HeLa cell lysate (20 µg/Lane) Lane 2: HeLa treated with 2μg/mL Tunicamycin for 8 hours cell lysate (20 µg/Lane) Lane 3: HeLa cell lysate (20 µg/Lane) Lane 4: HeLa treated with 250μM CoCl2 for 6 hours cell lysate (20 µg/Lane) Lane 5: NIH/3T3 cell lysate (20 µg/Lane) Lane 6: NIH/3T3 treated with 2μg/mL Tunicamycin for 8 hours cell lysate (20 µg/Lane) Predicted band size: 39 kDa Observed band size: 50 kDa Exposure time: 59 seconds; ECL: K1802; 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 (HA722796) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

ATF-4 was immunoprecipitated from 0.2 mg HeLa treated with 2μg/mL Tunicamycin for 8 hours cell lysate with HA722796 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA722796 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature. Lane 1: HeLa treated with 2μg/mL Tunicamycin for 8 hours cell lysate (input) Lane 2: HA722796 IP in HeLa treated with 2μg/mL Tunicamycin for 8 hours cell lysate Lane 3: Rabbit IgG instead of HA722796 in HeLa treated with 2μg/mL Tunicamycin for 8 hours cell lysate Blocking/Dilution buffer: 5% NFDM/TBST Exposure time: 40 seconds; ECL: K1802