Mmp 1

Western blot (WB) analysis for proteins isolated from wound tissue of animals with or without CD34⁺ therapy was performed by following the standard procedures. Primary antibodies used were for MMP1, α-SMA (from Santa Cruz, CA), MMP3, MMP9, MMP13, SM22α (all from Abcam, USA), β actin, and GAPDH (both from Cell Signaling, Beverly, MA). Mouse, rabbit IgG-HRP conjugated (Cell Signaling, Beverly, MA) and goat IgG-HRP conjugated (Santa Cruz, CA) secondary Abs were used and specific bands were detected using enzyme-linked chemiluminescences (Pierce, IL). Densitometric analysis of developed bands was performed by using UN-SCAN-IT (gel 6.1 version) software. Relative density was calculated using respective GAPDH/ β-actin bands.
In a separate experiment, fibroblasts were cultured under serum deprived (1% FBS) conditions. Total protein was extracted in lysis buffer containing protease and phosphatase inhibitors from 5 different conditions of fibroblast cultures, such as added MG132 (10 µM), CD34⁺ cells, CD34⁺ cells plus MG132, or MG132 plus SP 600125 (JNK Inhibitor II, 20 µM) (from Calbiochem, Darmstadt, Germany), with medium alone serving as a control at both 6 h and 12 h time points. Twenty micrograms of total proteins were tested by WB analysis for levels of c-Jun and GAPDH (all from Cell Signaling, Beverly, MA) following the above-mentioned techniques.