Increased LDH-A expression and activity in Taxol-resistant cells. A, Western blot was performed with an anti-LDH-A antibody of total cell extract from MDA-435, 435TR1 and 435TRP cells. The β-actin protein was used as a loading control. B, LDH activity in MDA-435, 435TR1 and 435TRP were examined. C, MDA-435 cells were treated with increasing concentrations of Taxol for 24 hrs. The cell lysates were prepared and Western blotting was carried out with antibodies against to LDH-A and β-actin. D, LDHA protein stability assay was performed in MDA-435 cells under the treatments of Taxol at 4 nM and CHX at 50 ug/ml followed by Western blotting assay to exam the protein expression level of LDHA at 0 and 8 hrs (top). The relative intensity of LDHA band was normalized to its β-actin loading (bottom). E, LDHA mRNA level was detected by real-time PCR under 2 nM Taxol in MDA-435 cells. The LDHA primers used for PCR are: forward, 5'-tgg agt gga atg aat gtt gc-3'; reverse: 5'-ata gcc cag gat gtg tag cc-3'. Columns, mean of three independent experiments; bars, SE. ***, P < 0.001.