Western blot analysis of Phospho-Histone H2A.X (S139) on different lysates with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/2;000 dilution. Lane 1: HeLa whole cell lysate Lane 2: HeLa treated with 20μM Etoposide for 2 hours whole cell lysate Lane 3: HeLa whole cell lysate Lane 4: HeLa treated with UV for 2 hours whole cell lysate Lane 5: NIH/3T3 whole cell lysate Lane 6: NIH/3T3 treated with 25μM Etoposide for 5 hours whole cell lysate Lysates/proteins at 20 µg/Lane. Predicted band size: 15 kDa Observed band size: 15/20 kDa Exposure time: 53 seconds; 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 (ET1602-2) at 1/2;000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100;000 dilution was used for 1 hour at room temperature.
Western blot analysis of HMGB1 on different lysates with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/50;000 dilution and competitor's antibody at 1/10;000 dilution. Lane 1: HepG2 cell lysate Lane 2: HeLa cell lysate Lane 3: HCT 116 cell lysate Lane 4: A549 cell lysate Lane 5: Jurkat cell lysate Lane 6: C2C12 cell lysate Lane 7: C6 cell lysate Lysates/proteins at 15 µg/Lane. Predicted band size: 25 kDa Observed band size: 25 kDa Exposure time: 21 seconds; 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 (ET1601-2) at 1/50;000 dilution and competitor's antibody at 1/10;000 dilution were 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.
Immunocytochemistry analysis of HeLa cells labeling HMGB1 with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/500 dilution and competitor's antibody at 1/250 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature; permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature; then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/500 dilution and competitor's antibody at 1/250 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488; HA1121) was used as the secondary antibody at 1/1;000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (M1305-2; red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594; HA1126) was used as the secondary antibody at 1/1;000 dilution.
Western blot analysis of HMGB1 on different lysates with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/20;000 dilution. Lane 1: HeLa cell lysate (20 µg/Lane) Lane 2: HCT 116 cell lysate (20 µg/Lane) Lane 3: A549 cell lysate (20 µg/Lane) Lane 4: HepG2 cell lysate (20 µg/Lane) Lane 5: Jurkat cell lysate (20 µg/Lane) Lane 6: Mouse thymus tissue lysate (20 µg/Lane) Lane 7: Rat spleen tissue lysate (30 µg/Lane) Predicted band size: 25 kDa Observed band size: 25 kDa Exposure time: 3 minutes 10 seconds; 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 (ET1601-2) at 1/20;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.
