Effect of increasing tensile strain on TIMP-1 mRNA and protein levels in mouse osteoblastic MC3T3-E1 cells. Cells were seeded at a density of 2 × 105 cells/well, cultured for 24 h, and were then loaded with (6%, 12%, 18%) or without (0%) tensile strain at the indicated percent elongation at 6 cycles/min using a Flexercell strain unit for 24 h. TIMP-1 mRNA and protein levels were determined by RT-PCR and Western blot analysis, respectively, as described in the Materials and methods. (A) Agarose gel electrophoresis of the RT-PCR products using primers specific for TIMP-1 or GAPDH. (B) TIMP-1 mRNA expression levels normalized to GAPDH mRNA levels. (C) Immunoblot analysis of TIMP-1 and GAPDH. (D) TIMP-1 protein expression levels normalized to GAPDH. The results shown are means ± SD of five independent experiments. Significant differences from the 0% culture are shown by ANOVA and Student's t test (*P < 0.05, ** P < 0.01).