The effect of in vitro heating on the distribution of nuclear matrix polypeptides in HeLa cells.
The in situ nuclear matrix was obtained from HeLa cells. After permeabilization with nonionic detergent, the resulting structures were incubated for 1 h at 37 degrees C to determine whether or not such an incubation might result in the redistribution of nuclear polypeptides which resisted extraction with buffers of high-ionic strength (1.6 M NaCl or 0.25 M (NH4)2SO4 as well as DNase I digestion. Using indirect immunofluorescence experiments and monoclonal antibodies we show that heating to 37 degrees C changes the distribution of a 160 kDa protein previously shown to be a component of the inner matrix network. On the other hand, a 125 kDa polypeptide was not affected at all by the incubation. Our results clearly indicate that the inclusion of a 37 degrees C incubation (for example during digestion with DNase I) in the protocol to obtain the in situ nuclear matrix can result in the formation of in vitro artifacts.
Martelli
A M
AM
Dipartimento di Morfologia Umana Normale, Trieste, Italy.
Bareggi
R
R
Riederer
B M
BM
Marugg
R A
RA
Narducci
P
P
D015153:Blotting, Western; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D015530:Nuclear Matrix / Q000737:chemistry*; D009687:Nuclear Proteins / Q000032:analysis*
D009687:Nuclear Proteins