All lanes : Anti-MCP1 antibody [EPR21025] (ab214819) at 1/1000 dilution

Lane 1 : Untreated THP-1 (human monocytic leukemia cell line) whole cell lysate
Lane 2 : THP-1 treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24 hours, then treated with 100ng/ml lipopolysaccharide (LPS) for 7 hours, then with 1 µg/ml Brefeldin A (BFA) added after 4 hours, whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 11 kDa
Observed band size: 11 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized THP-1 (human monocytic leukemia cell line) cells, untreated or treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24 hours, then treated with 100ng/ml lipopolysaccharide (LPS) for 7 hours, with 1 μg/ml Brefeldin A (BFA) added after 4 hours, labeling MCP1 with ab214819 at 1/50 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in THP-1 treated cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

MCP1 was immunoprecipitated from 0.35 mg of THP-1 (human monocytic leukemia cell line) treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1μg/ml Brefeldin A (BFA) for another 3h whole cell lysate with ab214819 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab214819 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1,000 dilution

Lane 1: THP-1 treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1μg/ml Brefeldin A (BFA) for another 3h whole cell lysate 10 µg (Input). 

Lane 2: ab214819 IP in THP-1 treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1μg/ml Brefeldin A (BFA) for another 3h whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab214819 in THP-1 treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1μg/ml Brefeldin A (BFA) for another 3h whole cell lysate.

Blocking and dilution buffer: 5% NFDM/TBST.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 0.1% Tween-20-permeabilized THP-1 (human monocytic leukemia cell line) cell line, treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1μg/ml Brefeldin A (BFA) for another 3h (Right) / Untreated control (Left) labeling MCP1 with ab214891 at 1/500 dilution. Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.