Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) cells transfected with a GFP-tagged mouse PCDH15 expression construct or GFP vector only, labeling PCDH15 with ab202560 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 594) (ab150080) secondary antibody at 1/1000 dilution (red).

Confocal image showing positive staining on HEK-293 cells transfected with a GFP-tagged mouse PCDH15 expression construct.

This mouse PCDH15 plasmid was kindly provided by our collaborator Dr. Yun Shi, Nanjing University MARC.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150115 (Alexa Fluor ^® 647 Goat anti-Mouse secondary) at 1/1000 dilution (white).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 594) (ab150080) at 1/1000 dilution.

 

All lanes : Anti-PCDH15 antibody [EPR19600] (ab202560) at 1/1000 dilution

Lane 1 : 293T (Human epithelial cell line from embryonic kidney) whole cell lysate transfected with an empty expression vector
Lane 2 : 293T whole cell lysate transfected with a DDDK-tagged mouse PCDH15 construct

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 215 kDa
Observed band size: 260 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature (PMID: 16481439).

The mouse PCDH15 plasmid was kindly provided by our collaborator Dr. Yun Shi, Nanjing University MARC.

WB detection of endogenous PCDH15 is not shown due to unavailability of appropriate testing material.