Hspb7

For analysis of protein levels by Western blot, whole tissue lysates were used from either the proteomic analysis or were prepared as described previously (34 (link)). Proteins were separated on precast SDS-PAGE 4–12% criterion gels (Bio-Rad) and transferred to polyvinylidene difluoride or nitrocellulose membranes. Site-specific antibodies directed to acetylated α-tubulin (Sigma, T7451), HSPA1 (Enzo Life Sciences, ADI-SPA-810), HSPA2 (Proteintech group, 66291-1), HSPB1 (Enzo Life Sciences, ADI-SPA-800), HSPB5 (Enzo Life Sciences, ADI-SPA-223), HSPB7 (abcam, ab150390), HSPD1 (Enzo Life Sciences, ADI-SPA-805), HSPA4 (Cell Signaling, 3303S), HSP90 (Cell Signaling, 4874S), α-tubulin (Sigma, T9026), tyrosinated tubulin (Sigma, T9028), detyrosinated tubulin (abcam, ab48389), and GAPDH (Cell Signaling, 2118S; Fitzgerald, 10R-G109a) were used to detect the proteins which were visualized with an enhanced chemiluminescence detection kit (Amersham) and scanned with Amersham Imager 600. Protein levels were determined by densitometric analysis and normalized to GAPDH.

Total cellular protein extracts were obtained with radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1% PMSF (Sigma) and 1% phosphatase inhibitor (Roche Applied Science). The concentration of protein was measured by the BCA protein assay kit (Thermo). Next, equal amounts of samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After being blocked with skimmed milk in TBST buffer (0.1 M Tris, 150 mM NaCl, and 0.1% Tween-20), the membranes were incubated overnight at 4 °C with primary antibodies against HSPB7 (Abcam, Cambridge, UK), RUNX2 (Abcam), OCN (Abcam), ERK1/2 (Cell Signaling Technology, Beverly, MA, USA), phosphorylated-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology), and GAPDH (HuaxingBio Science, Beijing, China). Subsequently, the membranes were washed three times with TBST and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies for 1 h at room temperature. The bands were visualized via the ECL Western Blot Kit (CoWin Biotech). The intensity of bands was quantified using ImageJ analysis software (http://rsb.info.nih.gov/ij/) and normalized to GAPDH.