Comparison of flow cytometry and western blotting to measure Hsp70.
The levels of constitutive and inducible forms of heat shock protein 70 (hsp73 and hsp72, respectively) through the cell cycle were measured in CHO cells by flow cytometry and Western blotting at various times after heating. Cells were labeled with antibody C92 (hsp72) or N27 (hsp73) and propidium iodide prior to analysis by flow cytometry. Cells were heated for 15 min at 45 degrees C, then analyzed from 3 to 36 h later. There was about a tenfold increase in hsp72 in early S phase cells beginning within 6 h after heating and these cells gradually cycled though S phase so by 36 h most of them had divided. When CHO cells were exposed to 10 microM sodium vanadate, an inhibitor of tyrosine phosphatase, for 24 h prior to heating, the induction of hsp72 in early S phase cells was almost completely inhibited. Heated cells did not express hsp73 in a cell-cycle-dependent manner. Hsp73 increased uniformly in all cells by 10 h after heating and sodium vanadate did not affect the expression. Quantitative comparisons of the relative levels of hsp72 and hsp73 measured by flow cytometry and Western blotting were in excellent agreement. Control and heated cells were labeled with Hoechst 33342 and sorted from G1, S, and G2/M phases and processed by Western blotting to verify the cell cycle dependent increase in hsp72 as measured by flow cytometry. Again there was excellent agreement between the Western blotting and flow cytometry results.
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Department of Radiological Health Sciences, Colorado State University, Fort Collins 80523, USA.
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D000818:Animals; D001562:Benzimidazoles / Q000737:chemistry; D015153:Blotting, Western / Q000379:methods*; D016466:CHO Cells; D002352:Carrier Proteins / Q000032:analysis*; D002453:Cell Cycle; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000032:analysis*; D006361:Heating; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D014638:Vanadates / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D001562:Benzimidazoles; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D014638:Vanadates; D014700:Verapamil; D017027:Protein Tyrosine Phosphatases; C017807:bisbenzimide ethoxide trihydrochloride