Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 cells labelling Coxsackie Adenovirus Receptor/hCAR with ab272711 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing membranous and cytoplasmic staining in HepG2 cell line. Negative control: K562 (PMID: 30450519) ab195887 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 488) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

All lanes : Anti-Coxsackie Adenovirus Receptor/hCAR antibody [EPR23305-44] (ab272711) at 1/1000 dilution

Lane 1 : LoVo (human colorectal adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 3 : PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : HaCaT (human skin keratinocyte) whole cell lysate
Lane 5 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 6 : BxPC-3 (human pancreas adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 40 kDa
Observed band size: 35-50 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The expression profile observed is consistent with what has been described in the literature, differently mobilized bands of coxsackie adenovirus receptor are attributed to different levels of glycosylation and the presence of isoforms. (PMID: 28074864, 11734628)

Negative control: PC-3 (PMID: 28074864) and K-562 (PMID: 30450519).

This blot was developed using a higher sensitivity ECL substrate. 

Exposure time: 3 minutes

All lanes : Anti-Coxsackie Adenovirus Receptor/hCAR antibody [EPR23305-44] (ab272711) at 1/1000 dilution

Lane 1 : Human brain tissue lysate
Lane 2 : Human liver tissue lysate
Lane 3 : Human heart tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP secondary antibody(HRP)(ab131366) at 1/1000 dilution

Predicted band size: 40 kDa
Observed band size: 45/35 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The expression profile observed is consistent with what has been described in the literature (PMID: 28074864, 11734628)

This blot was developed using a higher sensitivity ECL substrate. 

Exposure time: 125 seconds

Coxsackie Adenovirus Receptor/hCAR was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab272711 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab272711 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate 10 ug

Lane 2: ab272711 IP in HepG2 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab272711 in HepG2 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 102 seconds

 

Flow cytometric analysis of K-562 (Human chronic myelogenous leukemia lymphoblast, Left) / HepG2 (Human hepatocellular carcinoma epithelial cell, Right) cells labelling Coxsackie Adenovirus Receptor/hCAR with ab272711 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control: K-562 (PMID: 30450519)Gated on viable cells.