Untreated HUVEC cells, HUVEC cells (treated with 0.5 µg/ml LPS for 24 hours, then add 300 ng/ml protein transport inhibitor last 20 hours) were subjected to SDS PAGE followed by western blot with 21865-1-AP (IL-6 antibody) at dilution of 1:1000 incubated at room temperature for 1.5 hours.

1X10^6 HUVEC cells untreated (dashed lines), LPS and Brefeldin A treated (red) were intracellularly stained with 0.5 ug Anti-Human IL-6 (21865-1-AP) and CoraLite®488-Conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) at dilution 1:1000, or 0.5 ug Control Antibody (blue). Cells were fixed with 4% PFA and permeabilized with Intracellular Staining Permeabilization Wash Buffer.

Non-treated and protein transport inhibitor treated U-87 MG cells were subjected to SDS PAGE followed by western blot with 66146-1-Ig (IL-6 antibody) at dilution of 1:3000 incubated at room temperature for 1.5 hours.

Non-treated, LPS and protein transport inhibitor treated HUVEC cells were subjected to SDS PAGE followed by western blot with 66146-1-Ig (IL-6 antibody) at dilution of 1:3000 incubated at room temperature for 1.5 hours.

untreated, LPS and Brefeldin A treated RAW 264.7 cells were subjected to SDS PAGE followed by western blot with 26404-1-AP (IL-6 antibody) at dilution of 1:600 incubated at room temperature for 1.5 hours.

Recombinant human IL-6 (Cat.NO. HZ-1019) stimulates proliferation of hybridoma (Proteintech mouse anti-GST clone 3G12B10) in a dose-dependent manner (blue curve, refer to bottom X-left Y axis). The activity of human IL-6 (1 ng/mL HZ-1019) is neutralized by mouse anti-human IL-6 monoclonal antibody 69001-1-Ig at serial dose (red curve, refer to top X-right Y axis). The ND50 is typically 10-30 ng/mL. The NeutraControl mouse anti-human IL-6 monoclonal antibody 69501-1-Ig could recognize human IL-6 but could not neutralize human IL-6 activity in the range of 0.06 ng/mL-1000 ng/mL (green curve, refer to top X-right Y axis).

Indirect ELISA was carried out by coating recombinant Human IL-6 (Cat.NO. HZ-1019) at 70 ng/well followed by blocking and adding serial diluted IL-6 antibody 69001-1-Ig and 69501-1-Ig respectively. Signal was developed with TMB and stopped by H2SO4. Signal strength was measured by absorbance at 450 nm.

Standard curve of MP80002-1

Non-treated and protein transport inhibitor treated U-87 MG cells were subjected to SDS PAGE followed by western blot with 66146-1-Ig (IL-6 antibody) at dilution of 1:3000 incubated at room temperature for 1.5 hours. This data was developed using the same antibody clone with 66146-1-PBS in a different storage buffer formulation.

Non-treated, LPS and protein transport inhibitor treated HUVEC cells were subjected to SDS PAGE followed by western blot with 66146-1-Ig (IL-6 antibody) at dilution of 1:3000 incubated at room temperature for 1.5 hours. This data was developed using the same antibody clone with 66146-1-PBS in a different storage buffer formulation.

LPS and Brefeldin A treated RAW 264.7 cells were subjected to SDS PAGE followed by western blot with 29444-1-AP (IL-6 antibody) at dilution of 1:1000 incubated at room temperature for 1.5 hours.

HUVEC cells were subjected to SDS PAGE followed by western blot with 81077-1-RR (IL-6 antibody) at dilution of 1:5000 incubated at room temperature for 1.5 hours.

Protein transport inhibitor treated U-87 MG cells were subjected to SDS PAGE followed by western blot with 81077-1-RR (IL-6 antibody) at dilution of 1:5000 incubated at room temperature for 1.5 hours.