Journal: BMC Evolutionary Biology
Article Title: Functional test of PCDHB11, the most human-specific neuronal surface protein
Figure Lengend Snippet: Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and streptavidin-Alexa555, then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm
Article Snippet: For GFP detection, membranes were incubated with a rabbit polyclonal antibody (Life Technologies, A-11122) diluted 1:2000 in 50 mM Tris, 100 mM NaCl, 0.1 % Tween 20, pH 7.4, with 3 % bovine serum albumine (Sigma, St. Louis, MO) and a horse-radish peroxidase-conjugated anti-rabbit secondary antibody (Invitrogen, Waltham, MA), while for HA revelation, they were incubated with biotinylated 3 F10 rat monoclonal antibody (Roche Life Science, Indianapolis, IN) diluted 1:500 in the same buffer, and streptavidin-horseradish peroxidase (Invitrogen, Waltham, MA).
Techniques: Expressing, Transfection, Staining