Viral ORF57 expression is required to suppress the formation of SG during KSHV lytic infection and under arsenite stress.

ORF57 alone is sufficient to inhibit SG formation. HeLa cells transfected with an ORF57-Flag (pVM7) expressing vector or an empty vector (control) for 24 h were induced with 0.5 mM arsenite for 30 min for SG formation. The cells were stained for ORF57 (green), SG-specific TIA-1 (red) and PABPC1 (white) (ab21060) by each corresponding antibody. The nuclei were counterstained with Hoechst stain. Scale bar = 10 μm.

Anti-PABP antibody (ab21060) at 1 µg/ml + HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg/ml

Secondary
Goat polyclonal to Rabbit IgG (Alexa Fluor® 680) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 69 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

ab21060 detects a band of ~ 75 kDa in HeLa whole cell lysate. A 75 kDa band is also seen in Jurkat and A431 whole cell lysates (data not shown). PABP is reported to have run at an apparent molecular mass of 73 kDa on SDS-PAGE. We believe the band we see at 75 kDa represents PABP.

 

ICC/IF image of ab21060 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.
The cells were fixed in methanol (5 min) and incubated with the antibody (ab21060, 1 µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used to quench autofluorescence. 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor^® 594 WGA was used to label plasma membranes (red).

All lanes : Anti-PABP antibody (ab21060) at 1 µg/ml

Lane 1 : NIH/3T3 whole cell lysate (ab7179)
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 3 : Brain (Mouse) Tissue Lysate
Lane 4 : Mouse pancreas tissue lysate - total protein (ab29363)
Lane 5 : Testis (Mouse) Tissue Lysate - normal tissue
Lane 6 : Spinal Cord (Mouse) Tissue Lysate
Lane 7 : Ovary (Mouse) Tissue Lysate - normal tissue
Lane 8 : PC-12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
Lane 9 : Brain (Rat) Tissue Lysate - normal tissue

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 69 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?
Additional bands at: 110 kDa. We are unsure as to the identity of these extra bands.

ab21060 at 1/50 staining HeLa (Human epithelial cell line from cervix adenocarcinoma) cells by ICC/IF.

HeLa cells fixed for 10 minutes with 1.9 % formaldehyde in 2 % sucrose in PBS then permeabilized for 10 minutes with 0.5% NP40 in PBS-10% sucrose. The cells were then blocked in horse serum and  incubated with the antibody for 1 hour. An Alexa Fluor ^® 555 conjugated donkey anti-rabbit antibody was used as the secondary.

 

See Abreview

Immunocytochemical analysis of newborn porcine tracheal epithelial cells, labeling PABP with ab21060.

Cells were formaldehyde-fixed, permeabilized in cold methanol, and blocked with 5% horse serum in PBS for 40 minutes at 23°C. Immunostaining with primary anti-PABP diluted 1/250 for 12 hours at 4°C. 

See Abreview

Immunocytochemical analysis of rat OLN93 oligodendroglia cells, labeling PABP with ab21060.

Cells were paraformaldehyde-fixed, permeabilized with 0.1% Triton X-100, and blocked with 10% normal goat serum for 1 hour at 20°C. Immunostaining with anti-PABP diluted 1/200 overnight at 4°C.

See Abreview