Page 61 - Physico-Chemical Niche Conditions for Bone Cells

followed by 45 cycles of amplification at 95°C for 10 s, 56°C for 5 s, 72°C for 10 s, and 78°C for 5 s, after which melting curve analysis was performed. With LightCycler® software (version 1.2), crossing points were plotted versus the serial dilution of known concentrations of the internal standard. Values of target gene expression were normalized to PBGD (Forward: AGTGATGAAAGATGGGCAACT; Reverse: TCTGGACCATCTTCTTGCTGA) to obtain relative gene expression. RT-PCR was used to assess expression of the following genes:
Reverse: Forward: integrin -a5 Reverse: Forward: CTGCTGCTTATTGGACTCACCT; Reverse: GAGGAGGACCGTTGGTATATCTG), nesprin 2
paxillin-a (PXNA; Forward: CCTGGGCCA TGAACTTGAAA TC ),
CAGTCCGCAGCGAGTCA; paxillin-b (PXNB;
ACCAGGGAGAGATGAGCAGT; (ITGA51; Forward: TAGACAGCACCACCTTGCAG ),
Reverse: GGAAGGGACGGAGTCAGTG;
AGGCCCTGCATCTTGAAATCT), nesprin 1 (mNSP1var23;
(mNSP2; Forward:
TTGTCAAGGCAAAGTCACTCC),
TGCACCTGAGGAAGAGACAA;
obtained from 14 glass slides from 7 separate experiments (n = 7 for paxillin and integrin, n = 3 for nesprin 1, 2, and 4).
Protein Isolation and Western Blot
MC3T3-E1 osteoblasts were lysed in 600 μL isopropyl alcohol (IPA) buffer (Sigma-Aldrich). Total protein was measured using PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Blots were blocked in 5% BSA for 1 h, followed by incubation overnight at 4°C with primary antibodies against a-tubulin (Abcam) and pan-actin (Abcam). Then 1 h incubation with goat anti-rabbit IgG horseradish peroxidase (HRP) (Abcam) conjugated with horseradish peroxidase was performed, and membranes were monitored with a western light chemiluminescent detection system (Thermo Fisher Scientific). Protein bands were analyzed and quantified with Image J, and normalized to standard pan-actin levels. Data were obtained from eight glass slides from four separate experiments (n = 4).
Statistical Analysis
All data are presented as mean ± standard deviation (SD). Data were statistically tested with a t-test, and considered significant if p < 0.05. Statistical analysis was performed using International Business Machines Corporation (IBM®) Statistical Product and Service Solutions (SPSS®) Statistics version 21 software package (SPSS Inc., Chicago, IL, USA).
GAGACGCTCCTTCCTCTCAA;
and nesprin 4 (mNSP4;
Reverse:
Chapter 3
Reverse: CCGGAAGTTCAACCTCAACA ).
Forward: Data were
59
3